Secreted proteins

ABSTRACT

Various embodiments of the invention provide human secreted proteins(SECP) and polynucleotides which identify and encode SECP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SECP.

TECHNICAL FIELD

The invention relates to novel nucleic acids, secreted proteins encodedby these nucleic acids, and to the use of these nucleic acids andproteins in the diagnosis, treatment, and prevention of cellproliferative, autoimmune/inflammatory, cardiovascular, neurological,and developmental disorders. The invention also relates to theassessment of the effects of exogenous compounds on the expression ofnucleic acids and secreted proteins.

BACKGROUND OF THE INVENTION

Protein transport and secretion are essential for cellular function.Protein transport is mediated by a signal peptide located at the aminoterminus of the protein to be transported or secreted. The signalpeptide is comprised of about ten to twenty hydrophobic amino acidswhich target the nascent protein from the ribosome to a particularmembrane bound compartment such as the endoplasmic reticulum (ER).Proteins targeted to the ER may either proceed through the secretorypathway or remain in any of the secretory organelles such as the ER,Golgi apparatus, or lysosomes. Proteins that transit through thesecretory pathway are either secreted into the extracellular space orretained in the plasma membrane. Proteins that are retained in theplasma membrane contain one or more transmembrane domains, eachcomprised of about 20 hydrophobic amino acid residues. Secreted proteinsare generally synthesized as inactive precursors that are activated bypost-translational processing events during transit through thesecretory pathway. Such events include glycosylation, proteolysis, andremoval of the signal peptide by a signal peptidase. Other events thatmay occur during protein transport include chaperone-dependent unfoldingand folding of the nascent protein and interaction of the protein with areceptor or pore complex. Examples of secreted proteins with aminoterminal signal peptides are discussed below and include proteins withimportant roles in cell-to-cell signaling. Such proteins includetransmembrane receptors and cell surface markers, extracellular matrixmolecules, cytokines, hormones, growth and differentiation factors,enzymes, neuropeptides, vasomediators, cell surface markers, and antigenrecognition molecules. (Reviewed in Alberts, B. et al. (1994) MolecularBiology of The Cell, Garland Publishing, New York, N.Y., pp. 557-560,582-592.)

Cell surface markers include cell surface antigens identified onleukocytic cells of the immune system These antigens have beenidentified using systematic, monoclonal antibody (mAb)-based “shot gun”techniques. These techniques have resulted in the production of hundredsof mAbs directed against unknown cell surface leukocytic antigens. Theseantigens have been grouped into “clusters of differentiation” based oncommon immunocytochemical localization patterns in variousdifferentiated and undifferentiated leukocytic cell types. Antigens in agiven cluster are presumed to identify a single cell surface protein andare assigned a “cluster of differentiation” or “CD” designation. Some ofthe genes encoding proteins identified by CD antigens have been clonedand verified by standard molecular biology techniques. CD antigens havebeen characterized as both transmembrane proteins and cell surfaceproteins anchored to the plasma membrane via covalent attachment tofatty acid-containing glycolipids such as glycosylphosphatidylinositol(GPI). (Reviewed in Barclay, A. N. et al. (1995) The Leucocyte AntigenFacts Book, Academic Press, San Diego, Calif., pp. 17-20.)

Matrix proteins (MPs) are transmembrane and extracellular proteins whichfunction in formation, growth, remodeling, and maintenance of tissuesand as important mediators and regulators of the inflammatory response.The expression and balance of MPs may be perturbed by biochemicalchanges that result from congenital, epigenetic, or infectious diseases.In addition, MPs affect leukocyte migration, proliferation,differentiation, and activation in the immune response. MPs arefrequently characterized by the presence of one or more domains whichmay include collagen-like domains, EGF-like domains, immunoglobulin-likedomains, and fibronectin-like domains. In addition, MPs may be heavilyglycosylated and may contain an Arginine-Glycine-Aspartate (RGD)tripeptide motif which may play a role in adhesive interactions. MPsinclude extracellular proteins such as fibronectin, collagen, galectin,vitronectin and its proteolytic derivative somatomedin B; and celladhesion receptors such as cell adhesion molecules (CAMs), cadherins,and integrins. (Reviewed in Ayad, S. et al. (1994) The ExtracellularMatrix Facts Book, Academic Press, San Diego, Calif., pp. 2-16;Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M. D. and W. J.Nelson (1997) BioEssays 19:47-55.)

Mucins are highly glycosylated glycoproteins that are the majorstructural component of the mucus gel. The physiological functions ofmucins are cytoprotection, mechanical protection, maintenance ofviscosity in secretions, and cellular recognition. MUC6 is a humangastric mucin that is also found in gall bladder, pancreas, seminalvesicles, and female reproductive tract (Toribara, N. W. et al. (1997)J. Biol. Chem 272:16398-16403). The MUC6 gene has been mapped to humanchromosome 11 (Toribara, N. W. et al. (1993) J. Biol. Chem268:5879-5885). Hemomucin is a novel Drosophila surface mucin that maybe involved in the induction of antibacterial effector molecules(Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715).

Tuftelins are one of four different enamel matrix proteins that havebeen identified so far. The other three known enamel matrix proteins arethe amelogenins, enamelin and ameloblastin. Assembly of the enamelextracellular matrix from these component proteins is believed to becritical in producing a matrix competent to undergo mineral replacement(Paine, C. T. et al. (1998) Connect Tissue Res. 38:257-267). TuftelinmRNA has been found to be expressed in human ameloblastoma tumor, anon-mineralized odontogenic tumor (Deutsch, D. et al. (1998) Connect.Tissue Res. 39:177-184).

Olfactomedin-related proteins are extracellular matrix, secretedglycoproteins with conserved C-terminal motifs. They are expressed in awide variety of tissues and in a broad range of species, fromCaenorhabditis elegans to Homo sapiens. Olfactomedin-related proteinscomprise a gene family with at least 5 family members in humans. One ofthe five, TIGR/myocilin protein, is expressed in the eye and isassociated with the pathogenesis of glaucoma (Kulkarni, N. H. et al.(2000) Genet. Res. 76:41-50). Research by Yokoyama, M. et al. (1996; DNARes. 3:311-320) found a 135-amino acid protein, termed AMY, having 96%sequence identity with rat neuronal olfactomedin-releated ER localizedprotein in a neuroblastoma cell line cDNA library, suggesting anessential role for AMY in nerve tissue. Neuron-specificolfactomedin-related glycoproteins isolated from rat brain cDNAlibraries show strong sequence similarity with olfactomedin. Thissimilarity is suggestive of a matrix-related function of theseglycoproteins in neurons and neurosecretory cells (Danielson, P. E. etal. (1994) J. Neurosci. Res. 38:468-478).

Mac-2 binding protein is a 90-kD serum protein (90 K), a secretedglycoprotein isolated from both the human breast carcinoma cell lineSK-BR-3, and human breast milk. It specifically binds to a humanmacrophage-associated lectin, Mac-2. Structurally, the mature protein is567 amino acids in length and is proceeded by an 18-amino acid leader.There are 16 cysteines and seven potential N-linked glycosylation sites.The first 106 amino acids represent a domain very similar to an ancientprotein superfamily defined by a macrophage scavenger receptorcysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem.268:14245-14249). 90 K is elevated in the serum of subpopulations ofAIDS patients and is expressed at varying levels in primary tumorsamples and tumor cell lines. Ullrich, A. et al. (1994; J. Biol. Chem269:18401-18407) have demonstrated that 90 K stimulates host defensesystems and can induce interleukin-2 secretion. This immune stimulationis proposed to be a result of oncogenic transformation, viral infectionor pathogenic invasion (Ullrich et al., supra).

Semaphorins are a large group of axonal guidance molecules consisting ofat least 30 different members and are found in vertebrates,invertebrates, and even certain viruses. All semaphorins contain thesema domain which is approximately 500 amino acids in length.Neuropilin, a semaphorin receptor, has been shown to promote neuriteoutgrowth in vitro. The extracellular region of neuropilins consists ofthree different domains: CUB, discoidin, and MAM domains. The CUB andthe MAM motifs of neuropilin have been suggested to have roles inprotein-protein interactions and are thought to be involved in thebinding of semaphorins through the sema and the C-terminal domains(reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94).Plexins are neuronal cell surface molecules that mediate cell adhesionvia a homophilic binding mechanism in the presence of calcium ions.Plexins have been shown to be expressed in the receptors and neurons ofparticular sensory systems (Ohta, K. et al. (1995) Cell 14:1189-1199).There is evidence that suggests that some plexins function to controlmotor and CNS axon guidance in the developing nervous system. Plexins,which themselves contain complete semaphorin domains, may be both theancestors of classical semaphorins and binding partners for semaphorins(Winberg, ML. et al (1998) Cell 95:903-916).

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family ofclosely related glycoproteins of molecular weights of 72 KDa, 64 KDa, 62KDa, and 54 KDa. Together with the carcinoembryonic antigen, theycomprise a subfamily within the immunoglobulin superfamily (Plouzek, C.A. and J. Y. Chou, (1991) Endocrinology 129:950-958) Differentsubpopulations of PSG have been found to be produced by the trophoblastsof the human placenta, and the amnionic and chorionic membranes(Plouzek, C. A. et al. (1993) Placenta 14:277-285).

Torsion dystonia is an autosomal dominant movement disorder consistingof involuntary muscular contractions. The disorder has been linked to a3-base pair mutation in the DYT-1 gene, which encodes torsin A (Ozelius,L. J. et al. (1997) Nat. Genet. 17:4048). Torsin A bears significanthomology to the Hsp100/Clp family of ATPase chaperones, which areconserved in humans, rats, mice, and C. elegans. Strong expression ofDYT-1 in neuronal processes indicates a potential role for torsins insynaptic communication (Kustedjo, K. et al. (2000) J. Biol. Chem.275:27933-27939 and Konakova M. et al. (2001) Arch. Neurol. 58:921-927).

Autocrine motility factor (AMF) is one of the motility cytokinesregulating tumor cell migration; therefore identification of thesignaling pathway coupled with it has critical importance. Autocrinemotility factor receptor (AMFR) expression has been found to beassociated with tumor progression in thymoma (Ohta Y. et al. (2000) Int.J. Oncol. 17:259-264). AMFR is a cell surface glycoprotein of molecularweight 78 KDa

Hormones are secreted molecules that travel through the circulation andbind to specific receptors on the surface of, or within, target cells.Although they have diverse biochemical compositions and mechanisms ofaction, hormones can be grouped into two categories. One categoryincludes small lipophilic hormones that diffuse through the plasmamembrane of target cells, bind to cytosolic or nuclear receptors, andform a complex that alters gene expression. Examples of these moleculesinclude retinoic acid, thyroxine, and the cholesterol-derived steroidhormones such as progesterone, estrogen, testosterone, cortisol, andaldosterone. The second category includes hydrophilic hormones thatfunction by binding to cell surface receptors that transduce signalsacross the plasma membrane. Examples of such hormones include amino acidderivatives such as catecholamines (epinephrine, norepinephrine) andhistamine, and peptide hormones such as glucagon, insulin, gastrin,secretin, cholecystokinin, adrenocorticotropic hormone, folliclestimulating hormone, luteinizing hormone, thyroid stimulating hormone,and vasopressin. (See, for example, Lodish et al. (1995) Molecular CellBiology, Scientific American Books Inc., New York, N.Y., pp. 856-864.)

Pro-opiomelanocortin (POMC) is the precursor polypeptide ofcorticotropin (ACTH), a hormone synthesized by the anterior pituitarygland, which functions in the stimulation of the adrenal cortex. POMC isalso the precursor polypeptide of the hormone beta-lipotropin(beta-LPH). Each hormone includes smaller peptides with distinctbiological activities: alpha-melanotropin (alpha-MSH) andcorticotropin-like intermediate lobe peptide (CLIP) are formed fromACTH; gamma-lipotropin (gamma-LPH) and beta-endorphin are peptidecomponents of beta-LPH; while beta-MSH is contained within gamma-LPH.Adrenal insufficiency due to ACTH deficiency, resulting from a geneticmutation in exons 2 and 3 of POMC results in an endocrine disordercharacterized by early-onset obesity, adrenal insufficiency, and redhair pigmentation (Chretien, M. et al. (1979) Can. J. Biochem.57:1111-1121; Krude, H. et al. (1998) Nat. Genet. 19:155-157; OnlineMendelian Inheritance in Man (OMIM) 176830).

Growth and differentiation factors are secreted proteins which functionin intercellular communication. Some factors require oligomerization orassociation with membrane proteins for activity. Complex interactionsamong these factors and their receptors trigger intracellular signaltransduction pathways that stimulate or inhibit cell division, celldifferentiation, cell signaling, and cell motility. Most growth anddifferentiation factors act on cells in their local environment(paracrine signaling). There are three broad classes of growth anddifferentiation factors. The first class includes the large polypeptidegrowth factors such as epidermal growth factor, fibroblast growthfactor, transforming growth factor, insulin-like growth factor, andplatelet-derived growth factor. The second class includes thehematopoietic growth factors such as the colony stimulating factors(CSFs). Hematopoietic growth factors stimulate the proliferation anddifferentiation of blood cells such as B-lymphocytes, T-lymphocytes,erythrocytes, platelets, eosinophils, basophils, neutrophils,macrophages, and their stem cell precursors. The third class includessmall peptide factors such as bombesin, vasopressin, oxytocin,endothelin, transferrin, angiotensin II, vasoactive intestinal peptide,and bradykinin, which function as hormones to regulate cellularfunctions other than proliferation.

Growth and differentiation factors play critical roles in neoplastictransformation of cells in vitro and in tumor progression in vivo.Inappropriate expression of growth factors by tumor cells may contributeto vascularization and metastasis of tumors. During hematopoiesis,growth factor misregulation can result in anemias, leukemias, andlymphomas. Certain growth factors such as interferon are cytotoxic totumor cells both in vivo and in vitro. Moreover, some growth factors andgrowth factor receptors are related both structurally and functionallyto oncoproteins. In addition, growth factors affect transcriptionalregulation of both proto-oncogenes and oncosuppressor genes. (Reviewedin Pimentel, B. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor,Mich., pp. 1-9.)

The Slit protein, first identified in Drosophila, is critical in centralnervous system midline formation and potentially in nervous tissuehistogenesis and axonal pathfinding. Itoh et al. (1998; Brain Res. Mol.Brain Res. 62:175-186) have identified mammalian homologues of the slitgene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteinsare putative secreted proteins containing EGF-like motifs andleucine-rich repeats, both of which are conserved protein-proteininteraction domains. Slit-1, -2, and -3 mRNAs are expressed in thebrain, spinal cord, and thyroid, respectively (Itoh et al., supra). TheSlit family of proteins are indicated to be functional ligands ofglypican-1 in nervous tissue and it is suggested that their interactionsmay be critical in certain stages during central nervous systemhistogenesis (Liang, Y. et al. (1999) J. Biol. Chem. 274:17885-17892).

Neuropeptides and vasomediators (NP/VM) comprise a large family ofendogenous signaling molecules. Included in this family areneuropeptides and neuropeptide hormones such as bombesin, neuropeptideY, neurotensin, neuromedin N, melanocortins, opioids, galanin,somatostatin, tachykinins, urotensin II and related peptides involved insmooth muscle stimulation, vasopressin, vasoactive intestinal peptide,and circulatory system-borne signaling molecules such as angiotensin,complement, calcitonin, endothelins, formyl-methionyl peptides,glucagon, cholecystokinin and gastrin. NP/VMs can transduce signalsdirectly, modulate the activity or release of other neurotransmittersand hormones, and act as catalytic enzymes in cascades. The effects ofNP/VMs range from extremely brief to long-lasting. (Reviewed in Martin,C. R. et al. (1985) Endocrine Physiology, Oxford University Press, NewYork, N.Y., pp. 57-62.)

NP/VMs are involved in numerous neurological and cardiovasculardisorders. For example, neuropeptide Y is involved in hypertension,congestive heart failure, affective disorders, and appetite regulation.Somatostatin inhibits secretion of growth hormone and prolactin in theanterior pituitary, as well as inhibiting secretion in intestine,pancreatic acinar cells, and pancreatic beta-cells. A reduction insomatostatin levels has been reported in Alzheimer's disease andParkinson's disease. Vasopressin acts in the kidney to increase waterand sodium absorption, and in higher concentrations stimulatescontraction of vascular smooth muscle, platelet activation, and glycogenbreakdown in the liver. Vasopressin and its analogues are usedclinically to treat diabetes insipidus. Endothelin and angiotensin areinvolved in hypertension, and drugs, such as captopril, which reduceplasma levels of angiotensin, are used to reduce blood pressure (Watson,S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book,Academic Press, San Diego Calif., pp. 194; 252; 284; 55; 111).

Neuropeptides have also been shown to have roles in nociception (pain).Vasoactive intestinal peptide appears to play an important role inchronic neuropathic pain. Nociceptin, an endogenous ligand for for theopioid receptor-like 1 receptor, is thought to have a predominantlyanti-nociceptive effect, and has been shown to have analgesic propertiesin different animal models of tonic or chronic pain (Dickinson, T. andS. M. Fleetwood-Walker (1998) Trends Pharmacol. Sci. 19:346-348).

Other proteins that contain signal peptides include secreted proteinswith enzymatic activity. Such activity includes, for example,oxidoreductase/dehydrogenase activity, transferase activity, hydrolaseactivity, lyase activity, isomerase activity, or ligase activity. Forexample, matrix metalloproteinases are secreted hydrolytic enzymes thatdegrade the extracellular matrix and thus play an important role intumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. etal. (1995) Dev. Dyn. 202:388-396; Firestein, G. S. (1992) Curr. Opin.Rheumatol. 4:348-354; Ray, J. M. and W. G. Stetler-Stevenson (1994) Bur.Respir. J. 7:2062-2072; and Mignatti, P. and D. B. Riflin (1993)Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoAsynthetases which activate acetate for use in lipid synthesis or energygeneration (Luong, A. et al. (2000) J. Biol. Chem. 275:26458-26466). Theresult of acetyl-CoA synthetase activity is the formation of acetyl-CoAfrom acetate and CoA. Acetyl-CoA sythetases share a region of sequencesimilarity identified as the AMP-binding domain signature. Acetyl-CoAsynthetase has been shown to be associated with hypertension (Toh, H.(1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al. (1994)Hypertension 23:375-380).

A number of isomerases catalyze steps in protein folding,phototransduction, and various anabolic and catabolic pathways. Oneclass of isomerases is known as peptidyl-prolyl cis-trans isomerases(PPIases). PPIases catalyze the cis to trans isomerization of certainproline imidic bonds in proteins. Two families of PPIases are the FK506binding proteins (FKBPs), and cyclophilins (CyPs). FKBPs bind the potentimmunosuppressants FK506 and rapamycin, thereby inhibiting signalingpathways in T-cells. Specifically, the PPIase activity of FKBPs isinhibited by binding of FK506 or rapamycin. There are five members ofthe FKBP family which are named according to their calculated molecularmasses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and localized todifferent regions of the cell where they associate with differentprotein complexes (Coss, M. et al. (1995) J. Biol. Chem.270:29336-29341; Schreiber, S. L. (1991) Science 251:283-287).

The peptidyl-prolyl isomerase activity of CyP may be part of thesignaling pathway that leads to T-cell activation. CyP isomeraseactivity is associated with protein folding and protein trafficking, andmay also be involved in assembly/disassembly of protein complexes andregulation of protein activity. For example, in Drosophila, the CyPNinaA is required for correct localization of rhodopsins, while amammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that bindssteroid receptors. The mammalian CypA has been shown to bind the gagprotein from human immunodeficiency virus 1 (HIV-1), an interaction thatcan be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1activity, CypA may play an essential function in HIV-1 replication.Finally, Cyp40 has been shown to bind and inactivate the transcriptionfactor c-Myb, an effect that is reversed by cyclosporin. This effectimplicates CyPs in the regulation of transcription, transformation, anddifferentiation (Bergsma, D. J. et al (1991) J. Biol. Chem.266:23204-23214; Hunter, T. (1998) Cell 92:141-143; and Leverson, J. D.and S. A. Ness, (1998) Mol. Cell. 1:203-211).

Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) aremembers of a family of vitamin K-dependent single-pass integral membraneproteins. These proteins are characterized by an extracellular aminoterminal domain of approximately 45 amino acids rich in Gla. Theintracellular carboxyl terminal region contains one or two copies of thesequence PPXY, a motif present in a variety of proteins involved in suchdiverse cellular functions as signal transduction, cell cycleprogression, and protein turnover (Kulman, J. D. et al. (2001) Proc.Natl. Acad. Sci. USA 98:13701375). The process of post-translationalmodification of glutamic residues to form Gla is Vitamin K-dependentcarboxylation. Proteins which contain Gla include plasma proteinsinvolved in blood coagulation. These proteins are prothrombin, proteinsC, S, and Z, and coagulation factors VII, IX, and X. Osteocalcin(bone-Gla protein, BGP) and matrix Gla-protein (MGP) also contain Gla(Friedman, P. A. and C. T. Przysiecki (1987) Int. J. Biochem. 19:1-80;Vermeer, C. (1990) Biochem. J. 266:625-636).

Immunoglobulins

Antigen recognition molecules are key players in the sophisticated andcomplex immune systems which all vertebrates have developed to provideprotection from viral, bacterial, fungal, and parasitic infections. Akey feature of the immune system is its ability to distinguish foreignmolecules, or antigens, from “self” molecules. This ability is mediatedprimarily by secreted and transmembrane proteins expressed by leukocytes(white blood cells) such as lymphocytes, granulocytes, and monocytes.Most of these proteins belong to the immunoglobulin (Ig) superfamily,members of which contain one or more repeats of a conserved structuraldomain. This Ig domain is comprised of antiparallel β sheets joined by adisulfide bond in an arrangement called the Ig fold. The criteria for aprotein to be a member of the Ig superfamily is to have one or more Igdomains, which are regions of 70-110 amino acid residues in lengthhomologous to either Ig variable-like (V) or Ig constant-like (C)domains. Members of the Ig superfamily include antibodies (Ab), T cellreceptors (TCRs), class I and II major histocompatibility (MHC) proteinsand immune cell-specific surface markers such as the “cluster ofdifferentiation” or CD antigens, CD2, CD3, CD4, CD8, poly-Ig receptors,Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derivedgrowth factor receptor (PDGFR).

Ig domains (V and C) are regions of conserved amino acid residues thatgive a polypeptide a globular tertiary structure called animmunoglobulin (or antibody) fold, which consists of two approximatelyparallel layers of β-sheets. Conserved cysteine residues form anintrachain disulfide-bonded loop, 55-75 amino acid residues in length,which connects the two layers of β-sheets. Each β-sheet has three orfour anti-parallel β-strands of 5-10 amino acid residues. Hydrophobicand hydrophilic interactions of amino acid residues within the β-strandsstabilize the Ig fold (hydrophobic on inward facing amino acid residuesand hydrophilic on the amino acid residues in the outward facing portionof the strands). A V domain consists of a longer polypeptide than a Cdomain, with an additional pair of β-strands in the Ig fold.

A consistent feature of Ig superfamily genes is that each sequence of anIg domain is encoded by a single exon. It is possible that thesuperfamily evolved from a gene coding for a single Ig domain involvedin mediating cell-cell interactions. New members of the superfamily thenarose by exon and gene duplications. Modern Ig superfamily proteinscontain different numbers of V and/or C domains. Another evolutionaryfeature of this superfamily is the ability to undergo DNArearrangements, a unique feature retained by the antigen receptormembers of the family.

Many members of the Ig superfamily are integral plasma membrane proteinswith extracellular Ig domains. The hydrophobic amino acid residues oftheir transmembrane domains and their cytoplasmic tails are verydiverse, with little or no homology among Ig family members or to knownsignal-transducing structures. There are exceptions to this generalsuperfamily description. For example, the cytoplasmic tail of PDGFR hastyrosine kinase activity. In addition Thy-1 is a glycoprotein found onthymocytes and T cells. This protein has no cytoplasmic tail, but isinstead attached to the plasma membrane by a covalentglycophosphatidylinositol linkage.

Another common feature of many Ig superfamily proteins is theinteractions between Ig domains which are essential for the function ofthese molecules. Interactions between Ig domains of a multimeric proteincan be either homophilic or heterophilic (i.e., between the same ordifferent Ig domains). Antibodies are multimeric proteins which haveboth homophilic and heterophilic interactions between Ig domains.Pairing of constant regions of heavy chains forms the Fc region of anantibody and pairing of variable regions of light and heavy chains formthe antigen binding site of an antibody. Heterophilic interactions alsooccur between Ig domains of different molecules. These interactionsprovide adhesion between cells for significant cell-cell interactions inthe immune system and in the developing and mature nervous system.(Reviewed in Abbas, A. K. et al. (1991) Cellular and MolecularImmunology, W. B. Saunders Company, Philadelphia, Pa., pp. 142-145.)

Antibodies

MHC proteins are cell surface markers that bind to and present foreignantigens to T cells. MHC molecules are classified as either class I orclass II. Class I MHC molecules (MHC I) are expressed on the surface ofalmost all cells and are involved in the presentation of antigen tocytotoxic T cells. For example, a cell infected with virus will degradeintracellular viral proteins and express the protein fragments bound toMHC I molecules on the cell surface. The MHC I/antigen complex isrecognized by cytotoxic T-cells which destroy the infected cell and thevirus within. Class II MHC molecules are expressed primarily onspecialized antigen-presenting cells of the immune system, such asB-cells and macrophages. These cells ingest foreign proteins from theextracellular fluid and express MHC II/antigen complex on the cellsurface. This complex activates helper T-cells, which then secretecytokines and other factors that stimulate the immune response. MHCmolecules also play an important role in organ rejection followingtransplantation. Rejection occurs when the recipient's T-cells respondto foreign MHC molecules on the transplanted organ in the same way as toself MHC molecules bound to foreign antigen. (Reviewed in Alberts etal., supra, pp. 1229-1246.)

Antibodies are multimeric members of the Ig superfamily which are eitherexpressed on the surface of B-cells or secreted by B-cells into thecirculation. Antibodies bind and neutralize foreign antigens in theblood and other extracellular fluids. The prototypical antibody is atetramer consisting of two identical heavy polypeptide chains (H-chains)and two identical light polypeptide chains (L-chains) interlinked bydisulfide bonds. This arrangement confers the characteristic Y-shape toantibody molecules. Antibodies are classified based on their H-chaincomposition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, aredefined by the α, δ, ε, γ, and μ H-chain types. There are two types ofL-chains, κ and λ, either of which may associate as a pair with anyH-chain pair. IgG, the most common class of antibody found in thecirculation, is tetrameric, while the other classes of antibodies aregenerally variants or multimers of this basic structure.

H-chains and L-chains each contain an N-terminal variable region and aC-terminal constant region. The constant region consists of about 110amino acids in L-chains and about 330 or 440 amino acids in H-chains.The amino acid sequence of the constant region is nearly identical amongH- or L-chains of a particular class. The variable region consists ofabout 110 amino acids in both H- and L-chains. However, the amino acidsequence of the variable region differs among H- or L-chains of aparticular class. Within each H- or L-chain variable region are threehypervariable regions of extensive sequence diversity, each consistingof about 5 to 10 amino acids. In the antibody molecule, the H- andL-chain hypervariable regions come together to form the antigenrecognition site. (Reviewed in Alberts et al. supra, pp. 1206-1213;1216-1217.)

Both H-chains and L-chains contain the repeated Ig domains of members ofthe Ig superfamily. For example, a typical H-chain contains four Igdomains, three of which occur within the constant region and one ofwhich occurs within the variable region and contributes to the formationof the antigen recognition site. Likewise, a typical L-chain containstwo Ig domains, one of which occurs within the constant region and oneof which occurs within the variable region.

The immune system is capable of recognizing and responding to anyforeign molecule that enters the body. Therefore, the immune system mustbe armed with a full repertoire of antibodies against all potentialantigens. Such antibody diversity is generated by somatic rearrangementof gene segments encoding variable and constant regions. These genesegments are joined together by site-specific recombination which occursbetween highly conserved DNA sequences that flank each gene segment.Because there are hundreds of different gene segments, millions ofunique genes can be generated combinatorially. In addition, imprecisejoining of these segments and an unusually high rate of somatic mutationwithin these segments further contribute to the generation of a diverseantibody population.

Expression Profiling

Microarrays are analytical tools used in bioanalysis. A microarray has aplurality of molecules spatially distributed over, and stably associatedwith, the surface of a solid support. Microarrays of polypeptides,polynucleotides, and/or antibodies have been developed and find use in avariety of applications, such as gene sequencing, monitoring geneexpression, gene mapping, bacterial identification, drug discovery, andcombinatorial chemistry.

One area in particular in which microarrays find use is in geneexpression analysis. Array technology can provide a simple way toexplore the expression of a single polymorphic gene or the expressionprofile of a large number of related or unrelated genes. When theexpression of a single gene is examined, arrays are employed to detectthe expression of a specific gene or its variants. When an expressionprofile is examined, arrays provide a platform for identifying genesthat are tissue specific, are affected by a substance being tested in atoxicology assay, are part of a signaling cascade, carry outhousekeeping functions, or are specifically related to a particulargenetic predisposition, condition, disease, or disorder.

Steroids are a class of lipid-soluble molecules, including cholesterol,bile acids, vitamin D, and hormones, that share a common four-ringstructure based on cyclopentanoperhydrophenanthrene and that carrry outa wide variety of functions. Cholesterol, for example, is a component ofcell membranes that controls membrane fluidity. It is also a precursorfor bile acids which solubilize lipids and facilitate absorption in thesmall intestine during digestion. Vitamin D regulates the absorption ofcalcium in the small intestine and controls the concentration of calciumin plasma. Steroid hormones, produced by the adrenal cortex, ovaries,and testes, include glucocorticoids, mineralocorticoids, androgens, andestrogens. They control various biological processes by binding tointracellular receptors that regulate transcription of specific genes inthe nucleus. Glucocorticoids, for example, increase blood glucoseconcentrations by regulation of gluconeogenesis in the liver, increaseblood concentrations of fatty acids by promoting lipolysis in adiposetissues, modulate sensitivity to catcholamines in the central nervoussystem, and reduce inflammation. The principal mineralocorticoid,aldosterone, is produced by the adrenal cortex and acts on cells of thedistal tubules of the kidney to enhance sodium ion reabsorption.Androgens, produced by the interstitial cells of Leydig in the testis,include the male sex hormone testosterone, which triggers changes atpuberty, the production of sperm and maintenance of secondary sexualcharacteristics. Female sex hormones, estrogen and progesterone, areproduced by the ovaries and also by the placenta and adrenal cortex ofthe fetus during pregnancy. Estrogen regulates female reproductiveprocesses and secondary sexual characteristics. Progesterone regulateschanges in the endometrium during the menstrual cycle and pregnancy.

Steroid hormones are widely used for fertility control and inanti-inflammatory treatments for physical injuries and diseases such asarthritis, asthma, and auto-immune disorders. Progesterone, a naturallyoccurring progestin, is primarily used to treat amenorrhea, abnormaluterine bleeding, or as a contraceptive. Endogenous progesterone isresponsible for inducing secretory activity in the endometrium of theestrogen-primed uterus in preparation for the implantation of afertilized egg and for the maintenance of pregnancy. It is secreted fromthe corpus luteum in response to luteinizing hormone (LH). The primarycontraceptive effect of exogenous progestins involves the suppression ofthe midcycle surge of LH. At the cellular level, progestins diffusefreely into target cells and bind to the progesterone receptor. Targetcells include the female reproductive tract, the mammary gland, thehypothalamus, and the pituitary. Once bound to the receptor, progestinsslow the frequency of release of gonadotropin releasing hormone from thehypothalamus and blunt the pre-ovulatory LH surge, thereby preventingfollicular maturation and ovulation. Progesterone has minimal estrogenicand androgenic activity. Progesterone is metabolized hepatically topregnanediol and conjugated with glucuronic acid.

Medroxyprogesterone (MAH), also known as6α-methyl-17-hydroxyprogesterone, is a synthetic progestin with apharmacological activity about 15 times greater than progesterone. MAHis used for the treatment of renal and endometrial carcinomas,amenorrhea, abnormal uterine bleeding, and endometriosis associated withhormonal imbalance. MAH has a stimulatory effect on respiratory centersand has been used in cases of low blood oxygenation caused by sleepapnea, chronic obstructive pulmonary disease, or hypercapnia.

Mifepristone, also known as RU486, is an antiprogesterone drug thatblocks receptors of progesterone. It counteracts the effects ofprogesterone, which is needed to sustain pregnancy. Mifepristone inducesspontaneous abortion when administered in early pregnancy followed bytreatment with the prostaglandin, misoprostol. Further, studies showthat mifepristone at a substantially lower dose can be highly effectiveas a postcoital contraceptive when administered within five days afterunprotected intercourse, thus providing women with a “morning-afterpill” in case of contraceptive failure or sexual assault. Mifepristonealso has potential uses in the treatment of breast and ovarian cancersin cases in which tumors are progesterone-dependent. It interferes withsteroid-dependent growth of brain meningiomas, and may be useful intreatment of endometriosis where it blocks the estrogen-dependent growthof endometrial tissues. It may also be useful in treatment of uterinefibroid tumors and Cushing's Syndrome. Mifepristone binds toglucocorticoid receptors and interferes with cortisol binding.Mifepristone also may act as an anti-glucocorticoid and be effective fortreating conditions where cortisol levels are elevated such as AIDS,anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiplesclerosis, and Alzheimer's disease.

Danazol is a synthetic steroid derived from ethinyl testosterone.Danazol indirectly reduces estrogen production by lowering pituitarysynthesis of follicle-stimulating hormone and LH. Danazol also binds tosex hormone receptors in target tissues, thereby exhibiting anabolic,antiestrognic, and weakly androgenic activity. Danazol does not possessany progestogenic activity, and does not suppress normal pituitaryrelease of corticotropin or release of cortisol by the adrenal glands.Danazol is used in the treatment of endometriosis to relieve pain andinhibit endometrial cell growth. It is also used to treat fibrocysticbreast disease and hereditary angioedema.

Corticosteroids are used to relieve inflammation and to suppress theimmune response. They inhibit eosinophil, basophil, and airwayepithelial cell function by regulation of cytokines that mediate theinflammatory response. They inhibit leukocyte infiltration at the siteof inflammation, interfere in the function of mediators of theinflammatory response, and suppress the humoral immune response.Corticosteroids are used to treat allergies, asthma, arthritis, and skinconditions. Beclomethasone is a synthetic glucocorticoid that is used totreat steroid-dependent asthma, to relieve symptoms associated withallergic or nonallergic (vasomotor) rhinitis, or to prevent recurrentnasal polyps following surgical removal. The anti-inflammatory andvasoconstrictive effects of intranasal beclomethasone are 5000 timesgreater than those produced by hydrocortisone. Budesonide is acorticosteroid used to control symptoms associated with allergicrhinitis or asthma. Budesonide has high topical anti-inflammatoryactivity but low systemic activity. Dexamethasone is a syntheticglucocorticoid used in anti-inflammatory or immunosuppressivecompositions. It is also used in inhalants to prevent symptoms ofasthma. Due to its greater ability to reach the central nervous system,dexamethasone is usually the treatment of choice to control cerebraledema. Dexamethasone is approximately 20-30 times more potent thanhydrocortisone and 5-7 times more potent than prednisone. Prednisone ismetabolized in the liver to its active form, prednisolone, aglucocorticoid with anti-inflammatory properties. Prednisone isapproximately 4 times more potent than hydrocortisone and the durationof action of prednisone is intermediate between hydrocortisone anddexamethasone. Prednisone is used to treat allograft rejection, asthma,systemic lupus erythematosus, arthritis, ulcerative colitis, and otherinflammatory conditions. Betamethasone is a synthetic glucocorticoidwith antiinflammatory and immunosuppressive activity and is used totreat psoriasis and fungal infections, such as athlete's foot andringworm.

The anti-inflammatory actions of corticosteroids are thought to involvephospholipase A₂ inhibitory proteins, collectively called lipocortins.Lipocortins, in turn, control the biosynthesis of potent mediators ofinflammation such as prostaglandins and leukotrienes by inhibiting therelease of the precursor molecule arachidonic acid. Proposed mechanismsof action include decreased IgE synthesis, increased number ofβ-adrenergic receptors on leukocytes, and decreased arachidonic acidmetabolism. During an immediate allergic reaction, such as in chronicbronchial asthma, allergens bridge the IgE antibodies on the surface ofmast cells, which triggers these cells to release chemotacticsubstances. Mast cell influx and activation, therefore, is partiallyresponsible for the inflammation and hyperirritability of the oralmucosa in asthmatic patients. This inflammation can be retarded byadministration of corticosteroids.

Histological and molecular evaluation of breast tumors reveals that thedevelopment of breast cancer evolves through a multi-step processwhereby pre-malignant mammary epithelial cells undergo a relativelydefined sequence of events leading to tumor formation. An early event intumor development is ductal hyperplasia. Cells undergoing rapidneoplastic growth gradually progress to invasive carcinoma and becomemetastatic to the lung, bone, and potentially other organs. Severalvariables that may influence the process of tumor progression andmalignant transformation include genetic factors, environmental factors,growth factors, and hormones. Based on the complexity of this process,it is critical to study a population of human mammary epithelial cellsundergoing the process of malignant transformation, and to associatespecific stages of progression with phenotypic and molecularcharacteristics. We have compared primary breast epithelial cells(HMECs) to breast carcinoma lines at various stages of tumorprogression.

-   HMEC is a primary breast epithelial cell line isolated from a normal    donor.-   MCF-10A is a breast mammary gland (luminal ductal characteristics)    cell line that was isolated from a 36 year-old woman with    fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins,    epithelial sialomucins, and milkfat globule antigens. This cell    lines exhibits three-dimensional growth in collagen and forms domes    in confluent culture.-   MCF7 is a nonmalignant breast adenocarcinoma cell line isolated from    the pleural effusion of a 69-year-old female.-   MCF7 has retained characteristics of the mammary epithelium such as    the ability to process estradiol via cytoplasmic estrogen receptors    and the capacity to form domes in culture.-   T-47D is a breast carcinoma cell line isolated from a pleural    effusion obtained from a 54-year-old female with an infiltrating    ductal carcinoma of the breast.-   Sk-BR-3 is a breast adenocarcinoma cell line isolated from a    malignant pleural effusion of a 43-year-old female. It forms poorly    differentiated adenocarcinoma when injected into nude mice.-   BT-20 is a breast carcinoma cell line derived in vitro from cells    emigrating out of thin slices of the tumor mass isolated from a    74-year-old female.-   MDA-mb-231 is a breast tumor cell line isolated from the pleural    effusion of a 51-year old female. It forms poorly differentiated    adenocarcinoma in nude mice and ALS treated BALB/c mice. It also    expresses the Wnt3 oncogene, EGF, and TGF-α.-   MDA-mb-435S is a spindle shaped strain that evolved from the parent    line (435) as isolated in 1976 by R. Cailleau from the pleural    effusion of a 31-year-old female with metastatic, ductal    adenocarcinoma of the breast.

Osteosarcoma is the most common malignant bone tumor in children.Approximately 80% of patients present with non-metastatic disease. Afterthe diagnosis is made by an initial biopsy, treatment involves the useof 34 courses of neoadjuvant chemotherapy before definitive surgery,followed by post-operative chemotherapy. With currently availabletreatment regimens, approximately 30-40% of patients with non-metastaticdisease relapse after therapy. Currently, prognostic factor exists thatcan be used at the time of initial diagnosis to predict which patientswill have a high risk of relapse. The only significant prognostic factorpredicting the outcome in a patient with non-metastatic osteosarcoma isthe histopathologic response of the primary tumor resected at the timeof definitive surgery. The degree of necrosis in the primary tumor is areflection of the tumor response to neoadjuvant chemotherapy. A higherdegree of necrosis (good or favorable response) is associated with alower risk of relapse and a better outcome. Patients with a lower degreeof necrosis (poor or unfavorable response) have a much higher risk ofrelapse and poor outcome even after complete resection of the primarytumor. Unfortunately, poor outcome cannot be altered despitemodification of post-operative chemotherapy to account for theresistance of the primary tumor to neoadjuvant chemotherapy. Thus, thereis an urgent need to identify prognostic factors that can be used at thetime of diagnosis to recognize the subtypes of osteosarcomas that havevarious risks of relapse, so that more appropriate chemotherapy can beused at the outset to improve the outcome.

The most important function of adipose tissue is its ability to storeand release fat during periods of feeding and fasting. White adiposetissue is the major energy reserve in periods of excess energy use, andits primary purpose is mobilization during energy deprivation.Understanding how the various molecules regulate adiposity and energybalance in physiological and pathophysiological situations may lead tothe development of novel therapeutics for human obesity. Adipose tissueis also one of the important target tissues for insulin. Adipogenesisand insulin resistance in type II diabetes are linked and presentintriguing relations. Most patients with type II diabetes are obese andobesity in turn causes insulin resistance. Thiazolidinedione (TZD), afamily of drugs of peroxisome proliferation-activated receptor gamma(PPAR-γ) agonists, are a new class of antidiabetic agents that improveinsulin sensitivity and reduce plasma glucose and blood pressure insubjects with type II diabetes. TZD is also able to induce preadipocytesto differentiate into mature fat cells. The majority of research inadipocyte biology to date has been done using transformed mousepreadipocyte cell lines. It has been demonstrated that the culturecondition, which stimulates mouse preadipocyte differentiation isdifferent from that for inducing human primary preadipocytedifferentiation. In addition, primary cells are diploid and maytherefore reflect the in vivo context better than aneuploid cell lines.

Colon cancer is causally related to both genes and the environment.Several molecular pathways have been linked to the development of coloncancer, and the expression of key genes in any of these pathways may belost by inherited or acquired mutation or by hypermethylation. There isa particular need to identify genes for which changes in expression mayprovide an early indicator of colon cancer or a predisposition for thedevelopment of colon cancer.

For example, it is well known that abnormal patterns of DNA methylationoccur consistently in human tumors and include, simultaneously,widespread genomic hypomethylation and localized areas of increasedmethylation. In colon cancer in particular, it has been found that thesechanges occur early in tumor progression such as in premalignant polypsthat precede colon cancer. Indeed, DNA methyltransferase, the enzymethat performs DNA methylation, is significantly increased inhistologically normal mucosa from patients with colon cancer or thebenign polyps that precede cancer, and this increase continues duringthe progression of colonic neoplasms (Wafik, S. et al. (1991) Proc.Natl. Acad. Sci. USA 88:3470-3474). Increased DNA methylation occurs inG+C rich areas of genomic DNA termed “CpG islands” that are importantfor maintenance of an “open” transcriptional conformation around genes,and hypermethylation of these regions results in a “closed” conformationthat silences gene transcription. It has been suggested that thesilencing or downregulation of differentiation genes by such abnormalmethylation of CpG islands may prevent differentiation in immortalizedcells (Antequera, F. et al. (1990) Cell 62:503-514).

Familial Adenomatous Polyposis (FAP) is a rare autosomal dominantsyndrome that precedes colon cancer and is caused by an inheritedmutation in the adenomatous polyposis coli (APC) gene. FAP ischaracterized by the early development of multiple colorectal adenomasthat progress to cancer at a mean age of 44 years. The APC gene is apart of the APC-β-catenin-Tcf (T-cell factor) pathway. Impairment ofthis pathway results in the loss of orderly replication, adhesion, andmigration of colonic epithelial cells that results in the growth ofpolyps. A series of other genetic changes follow activation of theAPC-β-catenin-Tcf pathway and accompanies the transition from normalcolonic mucosa to metastatic carcinoma. These changes include mutationof the K-Ras proto-oncogene, changes in methylation patterns, andmutation or loss of the tumor suppressor genes p53 and Smad4/DPC4. Whilethe inheritance of a mutated APC gene is a rare event, the loss ormutation of APC and the consequent effects on the APC-β-catenin-Tcfpathway is believed to be central to the majority of colon cancers inthe general population.

Hereditary nonpolyposis Colorectal Cancer (HNPCC) is another inheritedautosomal dominant syndrome with a less well defined phenotype than FAP.HNPCC, which accounts for about 2% of colorectal cancer cases, isdistinguished by the tendency to early onset of cancer and thedevelopment of other cancers, particularly those involving theendometrium, urinary tract, stomach and biliary system. HNPCC resultsfrom the mutation of one or more genes in the DNA mis-match repair (MMR)pathway. Mutations in two human MMR genes, MSH2 and MLH1, are found in alarge majority of HNPCC families identified to date. The DNA MMR pathwayidentifies and repairs errors that result from the activity of DNApolymerase during replication. Furthermore, loss of MMR activitycontributes to cancer progression through accumulation of other genemutations and deletions, such as loss of the BAX gene which controlsapoptosis, and the TGFβ receptor II gene which controls cell growth.Because of the potential for irreparable damage to DNA in an individualwith a DNA MMR defect, progression to carcinoma is more rapid thanusual.

Although ulcerative colitis is a minor contributor to colon cancer,affected individuals have about a 20-fold increase in risk fordeveloping cancer. Progression is characterized by loss of the p53 genewhich may occur early, appearing even in histologically normal tissue.The progression of the disease from ulcerative colitis todysplasia/carcinoma without an intermediate polyp state suggests a highdegree of mutagenic activity resulting from the exposure ofproliferating cells in the colonic mucosa to the colonic contents.

Almost all colon cancers arise from cells in which the estrogen receptor(ER) gene has been silenced. The silencing of ER gene transcription isage related and linked to hypermethylation of the ER gene (Issa, J-P. J.et al. (1994) Nature Genetics 7:536-540). Introduction of an exogenousER gene into cultured colon carcinoma cells results in marked growthsuppression. The connection between loss of the ER protein in colonicepithelial cells and the consequent development of cancer has not beenestablished.

Clearly there are a number of genetic alterations associated with coloncancer and with the development and progression of the disease,particularly the downregulation or deletion of genes, that potentiallyprovide early indicators of cancer development, and which may also beused to monitor disease progression or provide possible therapeutictargets. The specific genes affected in a given case of colon cancerdepend on the molecular progression of the disease. Identification ofadditional genes associated with colon cancer and the precancerous statewould provide more reliable diagnostic patterns associated with thedevelopment and progression of the disease.

Prostate cancer is a common malignancy in men over the age of 50, andthe incidence increases with age. In the US, there are approximately132,000 newly diagnosed cases of prostate cancer and more than 33,000deaths from the disorder each year.

Once cancer cells arise in the prostate, they are stimulated bytestosterone to a more rapid growth. Thus, removal of the testes canindirectly reduce both rapid growth and metastasis of the cancer. Over95 percent of prostatic cancers are adenocarcinomas which originate inthe prostatic acini. The remaining 5 percent are divided betweensquamous cell and transitional cell carcinomas, both of which arise inthe prostatic ducts or other parts of the prostate gland.

As with most cancers, prostate cancer develops through a multistageprogression ultimately resulting in an aggressive, metastatic phenotype.The initial step in tumor progression involves the hyperproliferation ofnormal luminal and/or basal epithelial cells that become hyperplasticand evolve into early-stage tumors. The early-stage tumors are localizedin the prostate but eventually may metastasize, particularly to thebone, brain or lung. About 80% of these tumors remain responsive toandrogen treatment, an important hormone controlling the growth ofprostate epithelial cells. However, in its most advanced state, cancergrowth becomes androgen-independent and there is currently no knowntreatment for this condition.

A primary diagnostic marker for prostate cancer is prostate specificantigen (PSA). PSA is a tissue-specific serine protease almostexclusively produced by prostatic epithelial cells. The quantity of PSAcorrelates with the number and volume of the prostatic epithelial cells,and consequently, the levels of PSA are an excellent indicator ofabnormal prostate growth. Men with prostate cancer exhibit an earlylinear increase in PSA levels followed by an exponential increase priorto diagnosis. However, since PSA levels are also influenced by factorssuch as inflammation, androgen and other growth factors, some scientistsmaintain that changes in PSA levels are not useful in detectingindividual cases of prostate cancer.

Current areas of cancer research provide additional prospects formarkers as well as potential therapeutic targets for prostate cancer.Several growth factors have been shown to play a critical role in tumordevelopment, growth, and progression. The growth factors EpidermalGrowth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor GrowthFactor alpha (TGFα) are important in the growth of normal as well ashyperproliferative prostate epithelial cells, particularly at earlystages of tumor development and progression, and affect signalingpathways in these cells in various ways (Lin J et al. (1999) Cancer Res.59:2891-2897; Putz T et al. (1999) Cancer Res 59:227-233). The TGF-βfamily of growth factors are generally expressed at increased levels inhuman cancers and the high expression levels in many cases correlateswith advanced stages of malignancy and poor survival (Gold LI (1999)Crit Rev Oncog 10:303-360). Finally, there are human cell linesrepresenting both the androgen-dependent stage of prostate cancer(LNCaP) as well as the androgen-independent, hormone refractory stage ofthe disease (PC3 and DU-145) that have proven useful in studying geneexpression patterns associated with the progression of prostate cancer,and the effects of cell treatments on these expressed genes (Chung T D(1999) Prostate 15:199-207).

Alzheimer's disease is a progressive neurodegenerative disorder that ischaracterized by the formation of senile plaques and neurofibrillarytangles containing amyloid beta peptide. These plaques are found inlimbic and association cortices of the brain, including hippocampus,temporal cortices, cingulate cortex, amygdala, nucleus basalis and locuscaeruleus. Early in Alzheimer's pathology, physiological changes arevisible in the cingulate cortex (Minoshima, S. et al. (1997) Annals ofNeurology 42:85-94). In subjects with advanced Alzheimer's disease,accumulating plaques damage the neuronal architecture in limbic areasand eventually cripple the memory process.

Leukemias can be classified into four major categories, and all involvemalignant transformation of pluripotent stem cells. Acute leukemias,both lymphoblastic (ALL) and myeloid (AML) types, are characterized bythe presence of immature cells in the blood. Chronic leukemias, bothlymphocytic (CLL) and myelocytic (CML), are associated with mature,differentiated cells, but proportions of each cell type are abnormal.For example, CLL patients usually have clonal expansion of B celllymphocytes. CML patients often have granulocytes of all stages ofmaturity present in blood, bone marrow, and other organs. Monoclonalantibodies specific for B- and T-cells are helpful diagnostic tools, inaddition to histological analysis. Disease progresses as normalhematopoietic bone marrow is displaced by malignant cells. Cause hasbeen determined to be genetic in some cases, and chemical orradiation-induced in others.

There is a need in the art for new compositions, including nucleic acidsand proteins, for the diagnosis, prevention, and treatment of cellproliferative, autoimmune/inflammatory, cardiovascular, neurological,and developmental disorders.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides,secreted proteins, referred to collectively as “SECP” and individuallyas “SECP-1,” “SECP-2,” “SECP-3,” “SECP-4,” “SECP-5,” “SECP-6,” “SECP-7,”“SECP-8,” “SECP-9,” “SECP-10,” “SECP-11,” “SECP-12,” “SECP-13,”“SECP-14,” “SECP-15,” “SECP-16,” “SECP-17,” “SECP-18,” “SECP-19,”“SECP-20,” “SECP-21,” “SECP-22,” “SECP-23,” “SECP-24,” “SECP-25,”“SECP-26,” “SECP-27,” “SECP-28,” “SECP-29,” “SECP-30,” “SECP-31,”“SECP-32,”. “SECP-33,” “SECP-34,” “SECP-35,” “SECP-36,” “SECP-37,”“SECP-38,” “SECP-39,” “SECP-40,” “SECP-41,” “SECP-42,” “SECP-43,”“SECP-44,” “SECP-45,” “SECP-46,” “SECP-47,” “SECP-48,” “SECP-49,”“SECP-50,” “SECP-51,” “SECP-52,” “SECP-53,” “SECP-54,” “SECP-55,”“SECP-56,” “SECP-57,” “SECP-58,” “SECP-59,” “SECP-60,” “SECP-61,”“SECP-62,” “SECP-63,” “SECP-64,” “SECP-65,” “SECP-66,” “SECP-67,”“SECP-68,” “SECP-69,” “SECP-70,” “SECP-71,” “SECP-72,” “SECP-73,”“SECP-74,” “SECP-75,” “SECP-76,” “SECP-77,” “SECP-78,” “SECP-79,” and“SECP-80,” and methods for using these proteins and their encodingpolynucleotides for the detection, diagnosis, and treatment of diseasesand medical conditions. Embodiments also provide methods for utilizingthe purified secreted proteins and/or their encoding polynucleotides forfacilitating the drug discovery process, including determination ofefficacy, dosage, toxicity, and pharmacology. Related embodimentsprovide methods for utilizing the purified secreted proteins and/ortheir encoding polynucleotides for investigating the pathogenesis ofdiseases and medical conditions.

An embodiment provides an isolated polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-80, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-80. Another embodiment provides anisolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-80.

Still another embodiment provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ ID NO:1-80, c) a biologically active fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO: 1-80, andd) an immunogenic fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 1-80. Inanother embodiment, the polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO: 1-80. In an alternativeembodiment, the polynucleotide is selected from the group consisting ofSEQ ID NO:81-160.

Still another embodiment provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80. Another embodiment provides a celltransformed with the recombinant polynucleotide. Yet another embodimentprovides a transgenic organism comprising the recombinantpolynucleotide.

Another embodiment provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-80, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical or at least about 90% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:1-80, c) a biologically active fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO: 1-80, andd) an immunogenic fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 1-80. Themethod comprises a) culturing a cell under conditions suitable forexpression of the polypeptide, wherein said cell is transformed with arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide encoding the polypeptide, and b) recoveringthe polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specificallybinds to a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80.

Still yet another embodiment provides an isolated polynucleotideselected from the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:81-160, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:81-160, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In otherembodiments, the polynucleotide can comprise at least about 20, 30, 40,60, 80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:81-160, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:81-160, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) hybridizing the sample with a probe comprising at least 20contiguous nucleotides comprising a sequence complementary to saidtarget polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex. In a related embodiment, themethod can include detecting the amount of the hybridization complex. Instill other embodiments, the probe can comprise at least about 20, 30,40, 60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:81-160, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:81-160, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) amplifying said target polynucleotide or fragment thereofusing polymerase chain reaction amplification, and b) detecting thepresence or absence of said amplified target polynucleotide or fragmentthereof. In a related embodiment, the method can include detecting theamount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amountof a polypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-80, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ ID NO:1-80, c) a biologically active fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO: 1-80, andd) an immunogenic fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 1-80, and apharmaceutically acceptable excipient. In one embodiment, thecomposition can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80. Other embodiments provide a method oftreating a disease or condition associated with decreased or abnormalexpression of functional SECP, comprising administering to a patient inneed of such treatment the composition.

Yet another embodiment provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-80, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-80. The method comprises a)exposing a sample comprising the polypeptide to a compound, and b)detecting agonist activity in the sample. Another embodiment provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. Yet another embodiment providesa method of treating a disease or condition associated with decreasedexpression of functional SECP, comprising administering to a patient inneed of such treatment the composition.

Still yet another embodiment provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-80, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-80, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-80. The method comprises a)exposing a sample comprising the polypeptide to a compound, and b)detecting antagonist activity in the sample. Another embodiment providesa composition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. Yet another embodimentprovides a method of treating a disease or condition associated withoverexpression of functional SECP, comprising administering to a patientin need of such treatment the composition.

Another embodiment provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-80, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical or atleast about 90% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-80, c) a biologically active fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-80, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-80. The method comprises a) combining thepolypeptide with at least one test compound under suitable conditions,and b) detecting binding of the polypeptide to the test compound,thereby identifying a compound that specifically binds to thepolypeptide.

Yet another embodiment provides a method of screening for a compoundthat modulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-80, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-80, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-80. The method comprises a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Still yet another embodiment provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:81-160, the methodcomprising a) exposing a sample comprising the target polynucleotide toa compound, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.

Another embodiment provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:81-160, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical or at least about 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:81-160, iii) a polynucleotide having a sequence complementary to i),iv) a polynucleotide complementary to the polynucleotide of ii), and v)an RNA equivalent of i)-iv). Hybridization occurs under conditionswhereby a specific hybridization complex is formed between said probeand a target polynucleotide in the biological sample, said targetpolynucleotide selected from the group consisting of i) a polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:81-160, ii) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical or at leastabout 90% identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:81-160, iii) a polynucleotide complementary tothe polynucleotide of i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv).Alternatively, the target polynucleotide can comprise a fragment of apolynucleotide selected from the group consisting of i)-v) above; c)quantifying the amount of hybridization complex; and d) comparing theamount of hybridization complex in the treated biological sample withthe amount of hybridization complex in an untreated biological sample,wherein a difference in the amount of hybridization complex in thetreated biological sample is indicative of toxicity of the testcompound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide andpolypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog, and the PROTEOME database identificationnumbers and annotations of PROTEOME database homologs, for polypeptideembodiments of the invention. The probability scores for the matchesbetween each polypeptide and its homolog(s) are also shown.

Table 3 shows structural features of polypeptide embodiments, includingpredicted motifs and domains, along with the methods, algorithms, andsearchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide embodiments, along with selected fragments ofthe polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotideembodiments.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyzepolynucleotides and polypeptides, along with applicable descriptions,references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotideembodiments, along with allele frequencies in different humanpopulations.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described,it is understood that embodiments of the invention are not limited tothe particular machines, instruments, materials, and methods described,as these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a host cell” includes aplurality of such host cells, and a reference to “an antibody” is areference to one or more antibodies and equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with variousembodiments of the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

“SECP” refers to the amino acid sequences of substantially purified SECPobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of SECP. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of SECP either by directlyinteracting with SECP or by acting on components of the biologicalpathway in which SECP participates.

An “allelic variant” is an alternative form of the gene encoding SECP.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding SECP include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as SECP or a polypeptide with atleast one functional characteristic of SECP. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingSECP, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotideencoding SECP. The encoded protein may also be “altered,” and maycontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentSECP. Deliberate amino acid substitutions may be made on the basis ofone or more similarities in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of SECP isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valine; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” can refer to anoligopeptide, a peptide, a polypeptide, or a protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. Where “amino acid sequence” is recited to refer to a sequenceof a naturally occurring protein molecule, “amino acid sequence” andlike terms are not meant to limit the amino acid sequence to thecomplete native amino acid sequence associated with the recited proteinmolecule.

“Amplification” relates to the production of additional copies of anucleic acid. Amplification may be carried out using polymerase chainreaction (PCR) technologies or other nucleic acid amplificationtechnologies well known in the art.

The term “antagonist” refers to a molecule which inhibits Or attenuatesthe biological activity of SECP. Antagonists may include proteins suchas antibodies, anticalins, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition which modulates theactivity of SECP either by directly interacting with SECP or by actingon components of the biological pathway in which SECP participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind SECPpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a polynucleotide having a specificnucleic acid sequence. Antisense compositions may include DNA; RNA;peptide nucleic acid (PNA); oligonucleotides having modified backbonelinkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oligonucleotides having modified sugar groups suchas 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; oroligonucleotides having modified bases such as 5-methyl cytosine,2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may beproduced by any method including chemical synthesis or transcription.Once introduced into a cell, the complementary antisense moleculebase-pairs with a naturally occurring nucleic acid sequence produced bythe cell to form duplexes which block either transcription ortranslation. The designation “negative” or “minus” can refer to theantisense strand, and the designation “positive” or “plus” can refer tothe sense strand of a reference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic SECP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide” and a “compositioncomprising a given polypeptide” can refer to any composition containingthe given polynucleotide or polypeptide. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotides encoding SECP or fragments of SECP may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap(university of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions. Original Residue Conservative Substitution Ala Gly,Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn,Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, TyrSer Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu,Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an ally, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions(exons). Since an exon may represent a structural or functional domainof the encoded protein, new proteins may be assembled through the novelreassortment of stable substructures, thus allowing acceleration of theevolution of new protein functions.

A “fragment” is a unique portion of SECP or a polynucleotide encodingSECP which can be identical in sequence to, but shorter in length than,the parent sequence. A fragment may comprise up to the entire length ofthe defined sequence, minus one nucleotide/amino acid residue. Forexample, a fragment may comprise from about 5 to about 1000 contiguousnucleotides or amino acid residues. A fragment used as a probe, primer,antigen, therapeutic molecule, or for other purposes, may be at least 5,10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule. Forexample, a polypeptide fragment may comprise a certain length ofcontiguous amino acids selected from the first 250 or 500 amino acids(or first 25% or 50%) of a polypeptide as shown in a certain definedsequence. Clearly these lengths are exemplary, and any length that issupported by the specification, including the Sequence Listing, tables,and figures, may be encompassed by the present embodiments.

A fragment of SEQ ID NO:81-160 can comprise a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:81-160,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:81-160 can beemployed in one or more embodiments of methods of the invention, forexample, in hybridization and amplification technologies and inanalogous methods that distinguish SEQ ID NO:81-160 from relatedpolynucleotides. The precise length of a fragment of SEQ ID NO:81-160and the region of SEQ ID NO:81-160 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A fragment of SEQ ID NO: 1-80 is encoded by a fragment of SEQ IDNO:81-160. A fragment of SEQ ID NO: 1-80 can comprise a region of uniqueamino acid sequence that specifically identifies SEQ ID NO: 1-80. Forexample, a fragment of SEQ ID NO: 1-80 can be used as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO: 1-80. The precise length of a fragment of SEQ ID NO: 1-80 andthe region of SEQ ID NO: 1-80 to which the fragment corresponds can bedetermined based on the intended purpose for the fragment using one ormore analytical methods described herein or otherwise known in the art.

A “full length” polynucleotide is one containing at least a translationinitiation codon (e.g., methionine) followed by an open reading frameand a translation termination codon. A “full length” polynucleotidesequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, alternatively, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of identical residuematches between at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing one or more computer algorithms or programs known in the art ordescribed herein. For example, percent identity can be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program This program ispart of the LASERGENE software package, a suite of molecular biologicalanalysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described inHiggins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins,D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments ofpolynucleotide sequences, the default parameters are set as follows:Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The“weighted” residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms which can be used is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410),which is available from several sources, including the NCBI, Bethesda,Md., and on the internet at http://www.ncbi.nlm.nih.gov/BLAST/. TheBLAST software suite includes various sequence analysis programsincluding “blastn,” that is used to align a known polynucleotidesequence with other polynucleotide sequences from a variety ofdatabases. Also available is a tool called “BLAST 2 Sequences” that isused for direct pairwise comparison of two nucleotide sequences. “BLAST2 Sequences” can be accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters may be, for example:

-   -   Matrix: BLOSUM62    -   Reward for match: 1    -   Penalty for mismatch: −2    -   Open Gap: 5 and Extension Gap: 2 penalties    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 11    -   Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of identical residuematches between at least two polypeptide sequences aligned using astandardized algorithm. Methods of polypeptide sequence alignment arewell-known. Some alignment methods take into account conservative aminoacid substitutions. Such conservative substitutions, explained in moredetail above, generally preserve the charge and hydrophobicity at thesite of substitution, thus preserving the structure (and thereforefunction) of the polypeptide. The phrases “percent similarity” and “%similarity,” as applied to polypeptide sequences, refer to thepercentage of residue matches, including identical residue matches andconservative substitutions, between at least two polypeptide sequencesaligned using a standardized algorithm. In contrast, conservativesubstitutions are not included in the calculation of percent identitybetween polypeptide sequences.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

-   -   Matrix: BLOSUM62    -   Open Gap: 11 and Extension Gap: 1 penalties    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 3    -   Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific Specific hybridization complexes formunder permissive annealing conditions and remain hybridized after the“washing” step(s). The washing step(s) is particularly important indetermining the stringency of the hybridization process, with morestringent conditions allowing less non-specific binding, i.e., bindingbetween pairs of nucleic acid strands that are not perfectly matched.Permissive conditions for annealing of nucleic acid sequences areroutinely determinable by one of ordinary skill in the art and may beconsistent among hybridization experiments, whereas wash conditions maybe varied among experiments to achieve the desired stringency, andtherefore hybridization specificity. Permissive annealing conditionsoccur, for example, at 68° C. in the presence of about 6×SSC, about 1%(w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acids by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C₀t or R₀t analysis) or formed between one nucleic acid presentin solution and another nucleic acid immobilized on a solid support(e.g., paper, membranes, filters, chips, pins or glass slides, or anyother appropriate substrate to which cells or their nucleic acids havebeen fixed).

The words “insertion” and “addition” refer to changes in an amino acidor polynucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofSECP which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of SECPwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, antibodies, or other chemical compoundson a substrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, antibody, or other chemical compound having a unique anddefined position on a microarray.

The term “modulate” refers to a change in the activity of SECP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of SECP.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an SECP may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of SECP.

“Probe” refers to nucleic acids encoding SECP, their complements, orfragments thereof, which are used to detect identical, allelic orrelated nucleic acids. Probes are isolated oligonucleotides orpolynucleotides attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. “Primers” are short nucleic acids, usually DNAoligonucleotides, which may be annealed to a target polynucleotide bycomplementary base-pairing. The primer may then be extended along thetarget DNA strand by a DNA polymerase enzyme. Primer pairs can be usedfor amplification (and identification) of a nucleic acid, e.g., by thepolymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989; Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.), Ausubel, F. M. et al. (1999) Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, New York N.Y.), andInnis, M. et al. (1990, PCR Protocols. A Guide to Methods andApplications, Academic Press, San Diego Calif.). PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, hereby allowing selectionof primers that hybridize to either the most conserved or leastconserved regions of aligned nucleic acid sequences. Hence, this programis useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a nucleic acid that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA molecule, is composed of thesame linear sequence of nucleotides as the reference DNA molecule withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining SECP, nucleic acids encoding SECP, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably atleast about 75% free, and most preferably at least about 90% free fromother components with which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” or “expression profile” refers to the collectivepattern of gene expression by a particular cell type or tissue undergiven conditions at a given time.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. In another embodiment, the nucleicacid can be introduced by infection with a recombinant viral vector,such as a lentiviral vector (Lois, C. et al. (2002) Science295:868-872). The term genetic manipulation does not include classicalcross-breeding, or in vitro fertilization, but rather is directed to theintroduction of a recombinant DNA molecule. The transgenic organismscontemplated in accordance with the present invention include bacteria,cyanobacteria, fungi, plants and animals. The isolated DNA of thepresent invention can be introduced into the host by methods known inthe art, for example infection, transfection, transformation ortransconjugation. Techniques for transferring the DNA of the presentinvention into such organisms are widely known and provided inreferences such as Sambrook et al. (1989), supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 07, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotides that vary from onespecies to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity or sequencesimilarity to the particular polypeptide sequence over a certain lengthof one of the polypeptide sequences using blastp with the “BLAST 2Sequences” tool Version 2.0.9 (May 07, 1999) set at default parameters.Such a pair of polypeptides may show, for example, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity or sequence similarity over a certain defined lengthof one of the polypeptides.

The Invention

Various embodiments of the invention include new human secreted proteins(SECP), the polynucleotides encoding SECP, and the use of thesecompositions for the diagnosis, treatment, or prevention of cellproliferative, autoimmune/inflammatory, cardiovascular, neurological,and developmental disorders.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide embodiments of the invention. Each polynucleotide andits corresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown. Column 6 shows theIncyte ID numbers of physical, full length clones corresponding topolypeptide and polynucleotide embodiments. The full length clonesencode polypeptides which have at least 95% sequence identity to thepolypeptides shown in column 3.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database and the PROTEOME database. Columns 1 and 2 show thepolypeptide sequence identification number (Polypeptide SEQ ID NO:) andthe corresponding Incyte polypeptide sequence number (Incyte PolypeptideID) for polypeptides of the invention. Column 3 shows the GenBankidentification number (GenBank ID NO:) of the nearest GenBank homologand the PROTEOME database identification numbers (PROTEOME ID NO:) ofthe nearest PROTEOME database homologs. Column 4 shows the probabilityscores for the matches between each polypeptide and its homolog(s).Column 5 shows the annotation of the GenBank and PROTEOME databasehomolog(s) along with relevant citations where applicable, all of whichare expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Genetics Computer Group,Madison Wis. Column 6 shows amino acid residues comprising signaturesequences, domains, and motifs. Column 7 shows analytical methods forprotein structure/function analysis and in some cases, searchabledatabases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare secreted proteins. For example, SEQ ID NO: 16 is 71% identical, fromresidue M1 to residue D238, to human C1q-related factor (GenBank IDg3747097) as determined by the Basic Local Alignment Search Tool(BLAST). (See Table 2.) The BLAST probability score is 9.9e-91, whichindicates the probability of obtaining the observed polypeptide sequencealignment by chance. SEQ ID NO: 16 also contains a C1q domain asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS and MOTIFS analysesprovide further corroborative evidence that SEQ ID NO: 16 is aC1q-related complement factor. In an alternative example, SEQ ID NO:28is 41% identical, from residue M1 to residue L120, to Rattus norvegicusLy6C antigen (GenBank ID g205250) as determined by the Basic LocalAlignment Search Tool (BLAST). (See Table 2.) The BLAST probabilityscore is 5.0e-18, which indicates the probability of obtaining theobserved polypeptide sequence alignment by chance. SEQ ID NO:28 alsocontains a signal peptide and a u-Par/Ly-6 domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS analysis and from BLAST analysis of theDOMO database provide further corroborative evidence that SEQ ID NO:28is a secreted antigen. In an alternative example, SEQ ID NO:29 is 78%identical, from residue G66 to residue D129, to human PAP (pancreatitisassociated protein) homologous protein (GenBank ID g285971) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 8.5e-58, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. Pancreatitis associated protein I is a secretory stress proteinfirst characterized in pancreas during pancreatitis but also expressedin several tissues including hepatic, gastric, and colon cancer. Itsconcentration in serum can be significant. Exogenous pancreatitisassociated protein I can modify the adhesion and motility of normal andtransformed melanocytes, suggesting a potential interaction withmelanoma invasivity (Valery C et al (2001) J Invest Dermatol116(3):426-433.) SEQ ID NO:29 also contains a lectin C-type domain asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from MOTIFS, PROFILESCAN, BLIMPS,and further BLAST analyses provide corroborative evidence that SEQ IDNO:29 is a PAP homologous protein. In an alternative example, SEQ IDNO:45 is 78% identical, from residue G66 to residue D129, and 87%identical, from residue M1 to residue D65, to humanpancreatitis-associated protein (GenBank ID g482909) as determined bythe Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 8.5e-58, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:45 also contains a lectin C-type domain and a signal peptide asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS, PROFILESCAN, and MOTIFSanalyses and BLAST analyses of the PRODOM and DOMO databases providefurther corroborative evidence that SEQ ID NO:45 is a secretedlectin-related protein. In an alternative example, SEQ ID NO:58 is 98%identical, from residue D28 to residue L115 and 100% identical, fromresidue M1 to residue C27, to macaque epididymal secretory protein,ESP14.6 (GenBank ID g794071) as determined by the Basic Local AlignmentSearch Tool (BLAST). (See Table 2.) The BLAST probability score is2.6e-56, which indicates the probability of obtaining the observedpolypeptide sequence alignment by chance. SEQ ID NO:58 also contains aE1 family domain as determined by searching for statisticallysignificant matches in the hidden Markov model (HMM)-based PFAM databaseof conserved protein family domains. (See Table 3.) SEQ ID NO: 1-15, SEQID NO: 17-27, SEQ ID NO:3044, SEQ ID NO:46-57, and SEQ ID NO:59-80 wereanalyzed and annotated in a similar manner. The algorithms andparameters for the analysis of SEQ ID NO: 1-80 are described in Table 7.

As shown in Table 4, the full length polynucleotide embodiments wereassembled using cDNA sequences or coding (exon) sequences derived fromgenomic DNA, or any combination of these two types of sequences. Column1 lists the polynucleotide sequence identification number(Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotideconsensus sequence number (Incyte ID) for each polynucleotide of theinvention, and the length of each polynucleotide sequence in basepairs.Column 2 shows the nucleotide start (5′) and stop (3′) positions of thecDNA and/or genomic sequences used to assemble the full lengthpolynucleotide embodiments, and of fragments of the polynucleotideswhich are useful, for example, in hybridization or amplificationtechnologies that identify SEQ ID NO:81-160 or that distinguish betweenSEQ ID NO:81-160 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may referspecifically, for example, to Incyte cDNAs derived from tissue-specificcDNA libraries or from pooled cDNA libraries. Alternatively, thepolynucleotide fragments described in column 2 may refer to GenBankcDNAs or ESTs which contributed to the assembly of the full lengthpolynucleotides. In addition, the polynucleotide fragments described incolumn 2 may identify sequences derived from the ENSEMBL (The SangerCentre, Cambridge, UK) database (i.e., those sequences including thedesignation “ENST”). Alternatively, the polynucleotide fragmentsdescribed in column 2 may be derived from the NCBI RefSeq NucleotideSequence Records Database (i.e., those sequences including thedesignation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records(i.e., those sequences including the designation “NP”). Alternatively,the polynucleotide fragments described in column 2 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm. For example, a polynucleotide sequenceidentified as FL_XXXXXX_N_(1—)N_(2—)YYYYY_N_(3—)N₄ represents a“stitched” sequence in which XXXXXX is the identification number of thecluster of sequences to which the algorithm was applied, and YYYYY isthe number of the prediction generated by the algorithm, andN_(1,2,3...) , if present, represent specific exons that may have beenmanually edited during analysis (See Example V). Alternatively, thepolynucleotide fragments in column 2 may refer to assemblages of exonsbrought together by an “exon-stretching” algorithm For example, apolynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is a“stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may beused in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V). Type of analysis and/ Prefix or examples of programs GNN,GFG, Exon prediction from genomic ENST sequences using, for example,GENSCAN (Stanford University, CA, USA) or FGENES (Computer GenomicsGroup, The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis ofgenomic sequences. FL Stitched or stretched genomic sequences (seeExample V). INCY Full length transcript and exon prediction from mappingof EST sequences to the genome. Genomic location and EST compositiondata are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverageshown in Table 4 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotides which were assembled using Incyte cDNA sequences. Therepresentative cDNA library is the Incyte cDNA library which is mostfrequently represented by the Incyte cDNA sequences which were used toassemble and confirm the above polynucleotides. The tissues and vectorswhich were used to construct the cDNA libraries shown in Table 5 aredescribed in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found inpolynucleotide embodiments, along with allele frequencies in differenthuman populations. Columns 1 and 2 show the polynucleotide sequenceidentification number (SEQ ID NO:) and the corresponding Incyte projectidentification number (PID) for polynucleotides of the invention. Column3 shows the Incyte identification number for the EST in which the SNPwas detected (EST ID), and column 4 shows the identification number forthe SNP (SNP ID). Column 5 shows the position within the EST sequence atwhich the SNP is located (EST SNP), and column 6 shows the position ofthe SNP within the fill-length polynucleotide sequence (CB1 SNP). Column7 shows the allele found in the EST sequence. Columns 8 and 9 show thetwo alleles found at the SNP site. Column 10 shows the amino acidencoded by the codon including the SNP site, based upon the allele foundin the EST. Columns 11-14 show the frequency of allele 1 in fourdifferent human populations. An entry of n/d (not detected) indicatesthat the frequency of allele 1 in the population was too low to bedetected, while n/a (not available) indicates that the allele frequencywas not determined for the population.

The invention also encompasses SECP variants. A preferred SECP variantis one which has at least about 80%, or alternatively at least about90%, or even at least about 95% amino acid sequence identity to the SECPamino acid sequence, and which contains at least one functional orstructural characteristic of SECP.

Various embodiments also encompass polynucleotides which encode SECP. Ina particular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:81-160, which encodes SECP. The polynucleotide sequences of SEQ IDNO:81-160, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses variants of a polynucleotide encodingSECP. In particular, such a variant polynucleotide will have at leastabout 70%, or alternatively at least about 85%, or even at least about95% polynucleotide sequence identity to a polynucleotide encoding SECP.A particular aspect of the invention encompasses a variant of apolynucleotide comprising a sequence selected from the group consistingof SEQ ID NO:81-160 which has at least about 70%, or alternatively atleast about 85%, or even at least about 95% polynucleotide sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO:81-160. Any one of the polynucleotide variants describedabove can encode a polypeptide which contains at least one functional orstructural characteristic of SECP.

In addition, or in the alternative, a polynucleotide variant of theinvention is a splice variant of a polynucleotide encoding SECP. Asplice variant may have portions which have significant sequenceidentity to a polynucleotide encoding SECP, but will generally have agreater or lesser number of polynucleotides due to additions ordeletions of blocks of sequence arising from alternate splicing of exonsduring mRNA processing. A splice variant may have less than about 70%,or alternatively less than about 60%, or alternatively less than about50% polynucleotide sequence identity to a polynucleotide encoding SECPover its entire length; however, portions of the splice variant willhave at least about 70%, or alternatively at least about 85%, oralternatively at least about 95%, or alternatively 100% polynucleotidesequence identity to portions of the polynucleotide encoding SECP. Forexample, a polynucleotide comprising a sequence of SEQ ID NO: 153, apolynucleotide comprising a sequence of SEQ ID NO: 154, a polynucleotidecomprising a sequence of SEQ ID NO: 155, a polynucleotide comprising asequence of SEQ ID NO: 156, a polynucleotide comprising a sequence ofSEQ ID NO: 157, a polynucleotide comprising a sequence of SEQ ID NO:158, and a polynucleotide comprising a sequence of SEQ ID NO: 159 aresplice variants of each other; a polynucleotide comprising a sequence ofSEQ ID NO: 111 and a polynucleotide comprising a sequence of SEQ ID NO:116, are splice variants of each other; and a polynucleotide comprisinga sequence of SEQ ID NO: 160 and a polynucleotide comprising a sequenceof SEQ ID NO: 152 are splice variants of each other. Any one of thesplice variants described above can encode a polypeptide which containsat least one functional or structural characteristic of SECP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding SECP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring SECP, and all suchvariations are to be considered as being specifically disclosed.

Although polynucleotides which encode SECP and its variants aregenerally capable of hybridizing to polynucleotides encoding naturallyoccurring SECP under appropriately selected conditions of stringency, itmay be advantageous to produce polynucleotides encoding SECP or itsderivatives possessing a substantially different codon usage, e.g.,inclusion of non-naturally occurring codons. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encoding SECPand its derivatives without altering the encoded amino acid sequencesinclude the production of RNA transcripts having more desirableproperties, such as a greater half-life, than transcripts produced fromthe naturally occurring sequence.

The invention also encompasses production of polynucleotides whichencode SECP and SECP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic polynucleotide maybe inserted into any of the many available expression vectors and cellsystems using reagents well known in the art Moreover, syntheticchemistry may be used to introduce mutations into a polynucleotideencoding SECP or any fragment thereof.

Embodiments of the invention can also include polynucleotides that arecapable of hybridizing to the claimed polynucleotides, and, inparticular, to those having the sequences shown in SEQ ID NO:81-160 andfragments thereof, under various conditions of stringency (Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511). Hybridization conditions,including annealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Biosciences, Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Invitrogen, CarlsbadCalif.). Preferably, sequence preparation is automated with machinessuch as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.),PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST800 thermal cycler (Applied Biosystems). Sequencing is then carried outusing either the ABI 373 or 377 DNA sequencing system (AppliedBiosystems), the MEGABACE 1000 DNA sequencing system (AmershamBiosciences), or other systems known in the art. The resulting sequencesare analyzed using a variety of algorithms which are well known in theart (Ausubel et al., supra, ch 7; Meyers, R. A. (1995) Molecular Biologyand Biotechnology, Wiley VCH, New York N.Y., pp. 856853).

The nucleic acids encoding SECP may be extended utilizing a partialnucleotide sequence and employing various PCR-based methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector (Sarkar, G.(1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, usesprimers that extend in divergent directions to amplify unknown sequencefrom a circularized template. The template is derived from restrictionfragments comprising a known genomic locus and surrounding sequences(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method,capture PCR, involves PCR amplification of DNA fragments adjacent toknown sequences in human and yeast artificial chromosome DNA(Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In thismethod, multiple restriction enzyme digestions and ligations may be usedto insert an engineered double-stranded sequence into a region ofunknown sequence before performing PCR. Other methods which may be usedto retrieve unknown sequences are known in the art (Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotides or fragmentsthereof which encode SECP may be cloned in recombinant DNA moleculesthat direct expression of SECP, or fragments or functional equivalentsthereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other polynucleotides which encode substantially thesame or a functionally equivalent polypeptides may be produced and usedto express SECP.

The polynucleotides of the invention can be engineered using methodsgenerally known in the art in order to alter SECP-encoding sequences fora variety of purposes including, but not limited to, modification of thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, oligonucleotide-mediated site-directedmutagenesis may be used to introduce mutations that create newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of SECP, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding SECP may be synthesized,in whole or in part, using one or more chemical methods well known inthe art (Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser.7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).Alternatively, SECP itself or a fragment thereof may be synthesizedusing chemical methods known in the art. For example, peptide synthesiscan be performed using various solution-phase or solid-phase techniques(Creighton, T. (1984) Proteins, Structures and Molecular Properties, W HFreeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science269:202-204). Automated synthesis may be achieved using the ABI 431Apeptide synthesizer (Applied Biosystems). Additionally, the amino acidsequence of SECP, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990)Methods Enzymol. 182:392-421). The composition of the synthetic peptidesmay be confirmed by amino acid analysis or by sequencing. (Creighton,supra, pp. 28-53).

In order to express a biologically active SECP, the polynucleotidesencoding SECP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesencoding SECP. Such elements may vary in their strength and specificity.Specific initiation signals may also be used to achieve more efficienttranslation of polynucleotides encoding SECP. Such signals include theATG initiation codon and adjacent sequences, e.g. the Kozak sequence. Incases where a polynucleotide sequence encoding SECP and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used (Scharf,D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing polynucleotides encoding SECPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y., ch. 4, 8, and 16-17; Ausubel et al., supra, ch. 1, 3,and 15).

A variety of expression vector/host systems may be utilized to containand express polynucleotides encoding SECP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook,supra; Ausubel et al., supra; Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994) Proc. Natl.Acad. Sci. USA 91:32243227; Sandig, V. et al. (1996) Hum. Gene Ther.7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw HillYearbook of Science and Technology (1992) McGraw Hill, New York N.Y.,pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet 15:345-355).Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of polynucleotides to the targeted organ, tissue, or cellpopulation (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu,M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:63406344; Buller, R. M.et al. (1985) Nature 317:813-815; McGregor, D. P. et al. (1994) Mol.Immunol. 31:219-226; Verma, I. M. and N. Somia (1997) Nature389:239-242). The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotides encodingSECP. For example, routine cloning, subcloning, and propagation ofpolynucleotides encoding SECP can be achieved using a multifunctional E.coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1plasmid (Invitrogen). Ligation of polynucleotides encoding SECP into thevector's multiple cloning site disrupts the lacZ gene, allowing acalorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509). When large quantities of SECP are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofSECP may be used. For example, vectors containing the strong, inducibleSP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of SECP. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign polynucleotide sequences intothe host genome for stable propagation (Ausubel et al., supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al.(1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of SECP. Transcription ofpolynucleotides encoding SECP may be driven by viral promoters, e.g.,the 35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J.et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructscan be introduced into plant cells by direct DNA transformation orpathogen-mediated transfection (The McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,polynucleotides encoding SECP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses SECP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl.Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. SV40 or EBV-based vectors may alsobe used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997)Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems,stable expression of SECP in cell lines is preferred. For example,polynucleotides encoding SECP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ and apr⁻ cells, respectively (Wigler, M. et al. (1977) Cell11:223-232; Lowy, L et al. (1980) Cell 22:817-823). Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Wigler, M. et al.(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. etal. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, greenfluorescent proteins (GFP; Clontech), >glucuronidase and its substrateβ-glucuronide, or luciferase and its substrate luciferin may be used.These markers can be used not only to identify transformants, but alsoto quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes, C. A. (1995) MethodsMol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingSECP is inserted within a marker gene sequence, transformed cellscontaining polynucleotides encoding SECP can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding SECP under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding SECP andthat express SECP may be identified by a variety of procedures known tothose of skill in the art These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of SECPusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on SECPis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art (Hampton, R. et al. (1990)Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn.,Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding SECP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, polynucleotides encodingSECP, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as 17, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Biosciences, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with polynucleotides encoding SECP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeSECP may be designed to contain signal sequences which direct secretionof SECP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted polynucleotides or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding SECP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric SECPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of SECP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the SECP encodingsequence and the heterologous protein sequence, so that SECP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel et al. (supra, ch. 10 and 16). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

In another embodiment, synthesis of radiolabeled SECP may be achieved invitro using the TNT rabbit reticulocyte lysate or wheat germ extractsystem (Promega). These systems couple transcription and translation ofprotein-coding sequences operably associated with the 17, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, for example, ³⁵S-methionine.

SECP, fragments of SECP, or variants of SECP may be used to screen forcompounds that specifically bind to SECP. One or more test compounds maybe screened for specific binding to SECP. In various embodiments, 1, 2,3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened forspecific binding to SECP. Examples of test compounds can includeantibodies, anticalins, oligonucleotides, proteins (e.g., ligands orreceptors), or small molecules.

In related embodiments, variants of SECP can be used to screen forbinding of test compounds, such as antibodies, to SECP, a variant ofSECP, or a combination of SECP and/or one or more variants SECP. In anembodiment, a variant of SECP can be used to screen for compounds thatbind to a variant of SECP, but not to SECP having the exact sequence ofa sequence of SEQ ID NO:1-80. SECP variants used to perform suchscreening can have a range of about 50% to about 99% sequence identityto SECP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%,and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific bindingto SECP can be closely related to the natural ligand of SECP, e.g., aligand or fragment thereof, a natural substrate, a structural orfunctional mimetic, or a natural binding partner (Coligan, J. E. et al.(1991) Current Protocols in Immunology 1(2):Chapter 5). In anotherembodiment, the compound thus identified can be a natural ligand of areceptor SECP (Howard, A. D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specificbinding to SECP can be closely related to the natural receptor to whichSECP binds, at least a fragment of the receptor, or a fragment of thereceptor including all or a portion of the ligand binding site orbinding pocket. For example, the compound may be a receptor for SECPwhich is capable of propagating a signal, or a decoy receptor for SECPwhich is not capable of propagating a signal (Ashkenazi, A. and V. M.Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al.(2001) Trends Immunol. 22:328-336). The compound can be rationallydesigned using known techniques. Examples of such techniques includethose used to construct the compound etanercept (ENBREL; Amgen Inc.,Thousand Oaks Calif.), which is efficacious for treating rheumatoidarthritis in humans. Etanercept is an engineered p75 tumor necrosisfactor (TNF) receptor dimer linked to the Fc portion of human IgG₁(Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar or,alternatively, different specificities can be screened for specificbinding to SECP, fragments of SECP, or variants of SECP. The bindingspecificity of the antibodies thus screened can thereby be selected toidentify particular fragments or variants of SECP. In one embodiment, anantibody can be selected such that its binding specificity allows forpreferential identification of specific fragments or variants of SECP.In another embodiment, an antibody can be selected such that its bindingspecificity allows for preferential diagnosis of a specific disease orcondition having increased, decreased, or otherwise abnormal productionof SECP.

In an embodiment, anticalins can be screened for specific binding toSECP, fragments of SECP, or variants of SECP. Anticalins areligand-binding proteins that have been constructed based on a lipocalinscaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184;Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architectureof lipocalins can include a beta-barrel having eight antiparallelbeta-strands, which supports four loops at its open end. These loopsform the natural ligand-binding site of the lipocalins, a site which canbe re-engineered in vitro by amino acid substitutions to impart novelbinding specificities. The amino acid substitutions can be made usingmethods known in the art or described herein, and can includeconservative substitutions (e.g., substitutions that do not alterbinding specificity) or substitutions that modestly, moderately, orsignificantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to,stimulate, or inhibit SECP involves producing appropriate cells whichexpress SECP, either as a secreted protein or on the cell membrane.Preferred cells include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing SECP or cell membrane fractions which containSECP are then contacted with a test compound and binding, stimulation,or inhibition of activity of either SECP or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with SECP,either in solution or affixed to a solid support, and detecting thebinding of SECP to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to itsnatural ligand and/or to inhibit the binding of its natural ligand toits natural receptors. Examples of such assays include radio-labelingassays such as those described in U.S. Pat. No. 5,914,236 and U.S. Pat.No. 6,372,724. In a related embodiment, one or more amino acidsubstitutions can be introduced into a polypeptide compound (such as areceptor) to improve or alter its ability to bind to its natural ligands(Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). Inanother related embodiment, one or more amino acid substitutions can beintroduced into a polypeptide compound (such as a ligand) to improve oralter its ability to bind to its natural receptors (Cunningham, B. C.and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman,H. B. et al. (1991) J. Biol. Chem. 266:10982-10988).

SECP, fragments of SECP, or variants of SECP may be used to screen forcompounds that modulate the activity of SECP. Such compounds may includeagonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for SECPactivity, wherein SECP is combined with at least one test compound, andthe activity of SECP in the presence of a test compound is compared withthe activity of SECP in the absence of the test compound. A change inthe activity of SECP in the presence of the test compound is indicativeof a compound that modulates the activity of SECP. Alternatively, a testcompound is combined with an in vitro or cell-free system comprisingSECP under conditions suitable for SECP activity, and the assay isperformed. In either of these assays, a test compound which modulatesthe activity of SECP may do so indirectly and need not come in directcontact with the test compound. At least one and up to a plurality oftest compounds may be screened.

In another embodiment, polynucleotides encoding SECP or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:43234330). Transformed ES cells are identified and microinjectedinto mouse cell blastocysts such as those from the C57BL/6 mouse strain.The blastocysts are surgically transferred to pseudopregnant dams, andthe resulting chimeric progeny are genotyped and bred to produceheterozygous or homozygous strains. Transgenic animals thus generatedmay be tested with potential therapeutic or toxic agents.

Polynucleotides encoding SECP may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding SECP can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding SECP is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress SECP, e.g.,by secreting SECP in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of SECP and secreted proteins. Inaddition, examples of tissues expressing SECP can be found in Table 6and can also be found in Example III. Therefore, SECP appears to play arole in cell proliferative, autoimmune/inflammatory, cardiovascular,neurological, and developmental disorders. In the treatment of disordersassociated with increased SECP expression or activity, it is desirableto decrease the expression or activity of SECP. In the treatment ofdisorders associated with decreased SECP expression or activity, it isdesirable to increase the expression or activity of SECP.

Therefore, in one embodiment, SECP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of SECP. Examples ofsuch disorders include, but are not limited to, a cell proliferativedisorder such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, a cancer of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatorydisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; acardiovascular disorder such as congestive heart failure, ischemic heartdisease, angina pectoris, myocardial infarction, hypertensive heartdisease, degenerative valvular heart disease, calcific aortic valvestenosis, congenitally bicuspid aortic valve, mitral annularcalcification, mitral valve prolapse, rheumatic fever and rheumaticheart disease, infective endocarditis, nonbacterial thromboticendocarditis, endocarditis of systemic lupus erythematosus, carcinoidheart disease, cardiomyopathy, myocarditis, pericarditis, neoplasticheart disease, congenital heart disease, complications of cardiactransplantation, arteriovenous fistula, atherosclerosis, hypertension,vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicoseveins, thrombophlebitis and phlebothrombosis, vascular tumors, andcomplications of thrombolysis, balloon angioplasty, vascularreplacement, and coronary artery bypass graft surgery; a neurologicaldisorder such as epilepsy, ischemic cerebrovascular disease, stroke,cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington'sdisease, dementia, Parkinson's disease and other extrapyramidaldisorders, amyotrophic lateral sclerosis and other motor neurondisorders, progressive neural muscular atrophy, retinitis pigmentosa,hereditary ataxias, multiple sclerosis and other demyelinating diseases,bacterial and viral meningitis, brain abscess, subdural empyema,epidural abscess, suppurative intracranial thrombophlebitis, myelitisand radiculitis, viral central nervous system disease, prion diseasesincluding kuru, Creutzfeldt-Jakob disease, andGerstmnnn-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebefloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; and a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss.

In another embodiment, a vector capable of expressing SECP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofSECP including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified SECP in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of SECP including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofSECP may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of SECP including, butnot limited to, those listed above.

In a further embodiment, an antagonist of SECP may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of SECP. Examples of such disorders include, butare not limited to, those cell proliferative, autoimmune/inflammatory,cardiovascular, neurological, and developmental disorders describedabove. In one aspect, an antibody which specifically binds SECP may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissues whichexpress SECP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding SECP may be administered to a subject to treator prevent a disorder associated with increased expression or activityof SECP including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody,complementary sequence, or vector embodiments may be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy may be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of SECP may be produced using methods which are generallyknown in the art. In particular, purified SECP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind SECP. Antibodies to SECP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are generally preferred for therapeutic use.Single chain antibodies (e.g., from camels or llamas) may be potentenzyme inhibitors and may have advantages in the design of peptidemimetics, and in the development of immuno-adsorbents and biosensors(Muyldermans, S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with SECP or with any fragment or oligopeptidethereof which has immunogenic properties. Depending on the host species,various adjuvants may be used to increase immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to SECP have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein. Short stretches of SECP amino acids maybe fused with those of another protein, such as KLH, and antibodies tothe chimeric molecule may be produced.

Monoclonal antibodies to SECP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:3142; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. CellBiol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceSECP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837;Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for SECP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab)₂ fragments. Alternatively, Fab expression librariesmay be constructed to allow rapid and easy identification of monoclonalFab fragments with the desired specificity (Huse, W. D. et al. (1989)Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between SECP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering SECP epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for SECP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of SECP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple SECP epitopes, represents the average affinity,or avidity, of the antibodies for SECP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular SECP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theSECP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of SECP, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies. Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of SECP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable (Catty, supra; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding SECP,or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding SECP. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding SECP (Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press, Totawa N.J.).

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein (Slater, J. E. et al. (1998) J. Allergy Clin.Immunol. 102:469-475; Scanlon, K. J. et al. (1995) 9:1288-1296).Antisense sequences can also be introduced intracellularly through theuse of viral vectors, such as retrovirus and adeno-associated virusvectors (Miller, A. D. (1990) Blood 76:271; Ausubel et al., supra;Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Othergene delivery mechanisms include liposome-derived systems, artificialviral envelopes, and other systems known in the art (Rossi, J. J. (1995)Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res.25:2730-2736).

In another embodiment of the invention, polynucleotides encoding SECPmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475480; Bordignon, C. et al. (1995) Science270:470475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV,HCV); fungal parasites, such as Candida albicans and Paracoccidioidesbrasiliensis; and protozoan parasites such as Plasmodium falciparum andTrypanosoma cruzi). In the case where a genetic deficiency in SECPexpression or regulation causes disease, the expression of SECP from anappropriate population of transduced cells may alleviate the clinicalmanifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in SECP are treated by constructing mammalian expressionvectors encoding SECP and introducing these vectors by mechanical meansinto SECP-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of SECPinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT,PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF,PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). SECPmay be expressed using (i) a constitutively active promoter, (e.g., fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidinekinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and HL Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding SECP from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology 52:456467),or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). Theintroduction of DNA to primary cells requires modification of thesestandardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to SECP expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding SECP under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding SECP to cells which have one ormore genetic abnormalities with respect to the expression of SECP. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999; Annu. Rev. Nutr. 19:511-544) and Verma, I. M. and N. Somia (1997;Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding SECP to target cells which haveone or more genetic abnormalities with respect to the expression ofSECP. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing SECP to cells of the central nervoussystem, for which HSV has a tropism. The construction and packaging ofherpes-based vectors are well known to those with ordinary skill in theart. A replication-competent herpes simplex virus (HSV) type 1-basedvector has been used to deliver a reporter gene to the eyes of primates(Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of aHSV-1 virus vector has also been disclosed in detail in U.S. Pat. No.5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”),which is hereby incorporated by reference. U.S. Pat. No. 5,804,413teaches the use of recombinant HSV d92 which consists of a genomecontaining at least one exogenous gene to be transferred to a cell underthe control of the appropriate promoter for purposes including humangene therapy. Also taught by this patent are the construction and use ofrecombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSVvectors, see also Goins, W. et al. (1999; J. Virol. 73:519-532) and Xu,H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of clonedherpesvirus sequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding SECP to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for SECP into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of SECP-coding RNAs and the synthesis of high levels ofSECP in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:7483). The widehost range of alphaviruses will allow the introduction of SECP into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing, Mt Kisco N.Y., pp. 163-177).A complementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of RNA moleculesencoding SECP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be preparedby any method known in the art for the synthesis of nucleic acidmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA molecules encoding SECP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as 17 or SP6. Alternatively, these cDNA constructs thatsynthesize complementary RNA, constitutively or inducibly, can beintroduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding SECP. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased SECPexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding SECP may be therapeuticallyuseful, and in the treatment of disorders associated with decreased SECPexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding SECP may be therapeuticallyuseful.

At least one, and up to a plurality, of test compounds may be screenedfor effectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding SECP isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding SECP are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingSECP. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol.15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of SECP,antibodies to SECP, and mimetics, agonists, antagonists, or inhibitorsof SECP.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising SECP or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, SECP or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example SECP or fragments thereof, antibodies of SECP,and agonists, antagonists or inhibitors of SECP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000/μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind SECP may beused for the diagnosis of disorders characterized by expression of SECP,or in assays to monitor patients being treated with SECP or agonists,antagonists, or inhibitors of SECP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays or SECP include methods which utilizethe antibody and a label to detect SECP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring SECP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of SECP expression. Normal or standard values for SECPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to SECP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of SECP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding SECPmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotides, complementary RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantify geneexpression in biopsied tissues in which expression of SECP may becorrelated with disease. The diagnostic assay may be used to determineabsence, presence, and excess expression of SECP, and to monitorregulation of SECP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotides, including genomic sequences, encoding SECP orclosely related molecules may be used to identify nucleic acid sequenceswhich encode SECP. The specificity of the probe, whether it is made froma highly specific region, e.g., the 5′ regulatory region, or from a lessspecific region, e.g., a conserved motif, and the stringency of thehybridization or amplification will determine whether the probeidentifies only naturally occurring sequences encoding SECP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the SECP encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:81-160 or fromgenomic sequences including promoters, enhancers, and introns of theSECP gene.

Means for producing specific hybridization probes for polynucleotidesencoding SECP include the cloning of polynucleotides encoding SECP orSECP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotides encoding SECP may be used for the diagnosis of disordersassociated with expression of SECP. Examples of such disorders include,but are not limited to, a cell proliferative disorder such as actinickeratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis,primary thrombocythemia, and cancers including adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, a cancer of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; an autoimmune/inflammatory disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; a cardiovascular disorder such ascongestive heart failure, ischemic heart disease, angina pectoris,myocardial infarction, hypertensive heart disease, degenerative valvularheart disease, calcific aortic valve stenosis, congenitally bicuspidaortic valve, mitral annular calcification, mitral valve prolapse,rheumatic fever and rheumatic heart disease, infective endocarditis,nonbacterial thrombotic endocarditis, endocarditis of systemic lupuserythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,pericarditis, neoplastic heart disease, congenital heart disease,complications of cardiac transplantation, arteriovenous fistula,atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, and complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; and a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss. Polynucleotidesencoding SECP may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; in dipstick,pin, and multiformat ELISA-like assays; and in microarrays utilizingfluids or tissues from patients to detect altered SECP expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, polynucleotides encoding SECP may be used inassays that detect the presence of associated disorders, particularlythose mentioned above. Polynucleotides complementary to sequencesencoding SECP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantified and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of polynucleotides encoding SECP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of SECP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding SECP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding SECP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding SECP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding SECP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived frompolynucleotides encoding SECP may be used to detect single nucleotidepolymorphisms (SNPs). SNPs are substitutions, insertions and deletionsthat are a frequent cause of inherited or acquired genetic disease inhumans. Methods of SNP detection include, but are not limited to,single-stranded conformation polymorphism (SSCP) and fluorescent SSCP(fSSCP) methods. In SSCP, oligonucleotide primers derived frompolynucleotides encoding SECP are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (is SNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

SNPs may be used to study the genetic basis of human disease. Forexample, at least 16 common SNPs have been associated withnon-insulin-dependent diabetes mellitus. SNPs are also useful forexamining differences in disease outcomes in monogenic disorders, suchas cystic fibrosis, sickle cell anemia, or chronic granulomatousdisease. For example, variants in the mannose-binding lectin, MBL2, havebeen shown to be correlated with deleterious pulmonary outcomes incystic fibrosis. SNPs also have utility in pharmacogenomics, theidentification of genetic variants that influence a patient's responseto a drug, such as life-threatening toxicity. For example, a variationin N-acetyl transferase is associated with a high incidence ofperipheral neuropathy in response to the anti-tuberculosis drugisoniazid, while a variation in the core promoter of the ALOX5 generesults in diminished clinical response to treatment with an anti-asthmadrug that targets the 5-lipoxygenase pathway. Analysis of thedistribution of SNPs in different populations is useful forinvestigating genetic drift, mutation, recombination, and selection, aswell as for tracing the origins of populations and their migrations(Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. andZ. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr.Opin. Neurobiol. 11:637-641).

Methods which may also be used to quantify the expression of SECPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves(Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation ofmultiple samples may be accelerated by running the assay in ahigh-throughput format where the oligomer or polynucleotide of interestis presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotides described herein may be used as elementson a microarray. The microarray can be used in transcript imagingtechniques which monitor the relative expression levels of large numbersof genes simultaneously as described below. The microarray may also beused to identify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, SECP, fragments of SECP, or antibodies specificfor SECP may be used as elements on a microarray. The microarray may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time(Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No.5,840,484; hereby expressly incorporated by reference herein). Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471).If a test compound has a signature similar to that of a compound withknown toxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction of toxicity(see, for example, Press Release 00-02 from the National Institute ofEnvironmental Health Sciences, released Feb. 29, 2000, available athttp://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it isimportant and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In an embodiment, the toxicity of a test compound can be assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosedherein to analyze the proteome of a tissue or cell type. The termproteome refers to the global pattern of protein expression in aparticular tissue or cell type. Each protein component of a proteome canbe subjected individually to further analysis. Proteome expressionpatterns, or profiles, are analyzed by quantifying the number ofexpressed proteins and their relative abundance under given conditionsand at a given time. A profile of a cell's proteome may thus begenerated by separating and analyzing the polypeptides of a particulartissue or cell type. In one embodiment, the separation is achieved usingtwo-dimensional gel electrophoresis, in which proteins from a sample areseparated by isoelectric focusing in the first dimension, and thenaccording to molecular weight by sodium dodecyl sulfate slab gelelectrophoresis in the second dimension (Steiner and Anderson, supra).The proteins are visualized in the gel as discrete and uniquelypositioned spots, typically by staining the gel with an agent such asCoomassie Blue or silver or fluorescent stains. The optical density ofeach protein spot is generally proportional to the level of the proteinin the sample. The optical densities of equivalently positioned proteinspots from different samples, for example, from biological sampleseither treated or untreated with a test compound or therapeutic agent,are compared to identify any changes in protein spot density related tothe treatment. The proteins in the spots are partially sequenced using,for example, standard methods employing chemical or enzymatic cleavagefollowed by mass spectrometry. The identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least5 contiguous amino acid residues, to the polypeptide sequences ofinterest. In some cases, further sequence data may be obtained fordefinitive protein identification.

A proteomic profile may also be generated using antibodies specific forSECP to quantify the levels of SECP expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena,M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619;Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. etal. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat.No. 5,605,662). Various types of microarrays are well known andthoroughly described in Schena, M., ed. (1999; DNA Microarrays: APractical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encodingSECP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat.Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acidsequences may be used to develop genetic linkage maps, for example,which correlate the inheritance of a disease state with the inheritanceof a particular chromosome region or restriction fragment lengthpolymorphism (RFLP) (Lander, E. S. and D. Botstein (1986) Proc. Natl.Acad. Sci. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers,supra, pp. 965-968). Examples of genetic map data can be found invarious scientific journals or at the Online Mendelian Inheritance inMan (OMIM) World Wide Web site. Correlation between the location of thegene encoding SECP on a physical map and a specific disorder, or apredisposition to a specific disorder, may help define the region of DNAassociated with that disorder and thus may further positional cloningefforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation (Gatti, R. A. et al. (1988) Nature 336:577-580). Thenucleotide sequence of the instant invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, SECP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between SECPand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest (Geysen, et al. (1984) PCT application WO84/03564). In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate. The test compounds are reacted with SECP, orfragments thereof, and washed. Bound SECP is then detected by methodswell known in the art. Purified SECP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding SECP specificallycompete with a test compound for binding SECP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with SECP.

In additional embodiments, the nucleotide sequences which encode SECPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentionedabove and below, including U.S. Ser. No. 60/313,249, including U.S. Ser.No. 60/314,752, U.S. Ser. No. 60/317,824, U.S. Ser. No. 60/317,818, U.S.Ser. No. 60/324,586, U.S. Ser. No. 60/362,439, U.S. Ser. No. 60/357,002,U.S. Ser. No. 60/343,980, U.S. Ser. No. 60/334,229, U.S. Ser. No.60/366,041, U.S. Ser. No. 60/376,988, and U.S. Ser. No. 60/324,040 areexpressly incorporated by reference herein.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.). Some tissues werehomogenized and lysed in guanidinium isothiocyanate, while others werehomogenized and lysed in phenol or in a suitable mixture of denaturants,such as TRIZOL (Invitrogen), a monophasic solution of phenol andguanidine isothiocyanate. The resulting lysates were centrifuged overCsCl cushions or extracted with chloroform. RNA was precipitated fromthe lysates with either isopropanol or sodium acetate and ethanol, or byother routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+ RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Invitrogen), using the recommendedprocedures or similar methods known in the art (Ausubel et al., supra,ch. 5). Reverse transcription was initiated using oligo d(T) or randomprimers. Synthetic oligonucleotide adapters were ligated to doublestranded cDNA, and the cDNA was digested with the appropriaterestriction enzyme or enzymes. For most libraries, the cDNA wassize-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, orSEPHAROSE CL4B column chromatography (Amersham Biosciences) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid(Invitrogen), PcDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMVplasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICISplasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto Calif.), pRARE(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.Recombinant plasmids were transformed into competent E. coli cellsincluding XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B,or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWEL 8 Plasmid, QIAWEL 8 Plus Plasmid, QIAWELL8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmidpurification kit from QIAGEN. Following precipitation, plasmids wereresuspended in 0.1 ml of distilled water and stored, with or withoutlyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamBiosciences or supplied in ABI sequencing kits such as the ABI PRISMBIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (Ausubel et al., supra, ch. 7). Someof the cDNA sequences were selected for extension using the techniquesdisclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM;PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus,Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, PaloAlto Calif.); hidden Markov model (HMM)-based protein family databasessuch as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic AcidsRes. 29:41-43); and M-based protein domain databases such as SMART(Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;Letunic, L et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is aprobabilistic approach which analyzes consensus primary structures ofgene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct.Biol. 6:361-365.) The queries were performed using programs based onBLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences wereassembled to produce full length polynucleotide sequences.Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,stretched sequences, or Genscan-predicted coding sequences (see ExamplesIV and V) were used to extend Incyte cDNA assemblages to full length.Assembly was performed using programs based on Phred, Phrap, and Consed,and cDNA assemblages were screened for open reading frames usingprograms based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length polypeptide sequences. Alternatively, a polypeptide maybegin at any of the methionine residues of the full length translatedpolypeptide. Full length polypeptide sequences were subsequentlyanalyzed by querying against databases such as the GenBank proteindatabases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein familydatabases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domaindatabases such as SMART. Full length polynucleotide sequences are alsoanalyzed using MACDNASIS PRO software (MiraiBio Inc., Alameda Calif.)and LASERGENE software (DNASTAR). Polynucleotide and polypeptidesequence alignments are generated using default parameters specified bythe CLUSTAL algorithm as incorporated into the MEGALIGN multisequencealignment program (DNASTAR), which also calculates the percent identitybetween aligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:81-160.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative secreted proteins were initially identified by running theGenscan gene identification program against public genomic sequencedatabases (e.g., gbpri and gbhtg). Genscan is a general-purpose geneidentification program which analyzes genomic DNA sequences from avariety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode secreted proteins, the encoded polypeptides wereanalyzed by querying against PFAM models for secreted proteins.Potential secreted proteins were also identified by homology to IncytecDNA sequences that had been annotated as secreted proteins. Theseselected Genscan-predicted sequences were then compared by BLASTanalysis to the genpept and gbpri public databases. Where necessary, theGenscan-predicted sequences were then edited by comparison to the topBLAST hit from genpept to correct errors in the sequence predicted byGenscan, such as extra or omitted exons. BLAST analysis was also used tofind any Incyte cDNA or public cDNA coverage of the Genscan-predictedsequences, thus providing evidence for transcription. When Incyte cDNAcoverage was available, this information was used to correct or confirmthe Genscan predicted sequence. Full length polynucleotide sequenceswere obtained by assembling Genscan-predicted coding sequences withIncyte cDNA sequences and/or public cDNA sequences using the assemblyprocess described in Example III. Alternatively, full lengthpolynucleotide sequences were derived entirely from edited or uneditedGenscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data

“Stitched” Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of SECP Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:81-160 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm Sequences from these databases that matched SEQ ID NO:81-160were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Généthon were used to determine if any of theclustered sequences had been previously mapped. Inclusion of a mappedsequence in a cluster resulted in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Généthon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook, supra, ch. 7; Ausubel et al.,supra, ch. 4).

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in databases such as GenBank or LIFESEQ(Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{{BLAST}\quad{Score} \times {Percent}\quad{Identity}}{5 \times {minimum}\quad\left\{ {{{length}\left( {{Seq}.\quad 1} \right)},{{length}\left( {{Seq}.\quad 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and 4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotides encoding SECP are analyzed with respectto the tissue sources from which they were derived. For example, somefull length sequences are assembled, at least in part, with overlappingIncyte cDNA sequences (see Example III). Each cDNA sequence is derivedfrom a cDNA library constructed from a human tissue. Each human tissueis classified into one of the following organ/tissue categories:cardiovascular system; connective tissue; digestive system; embryonicstructures; endocrine system; exocrine glands; genitalia, female;genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding SECP. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of SECP Encoding Polynucleotides

Full length polynucleotides are produced by extension of an appropriatefragment of the full length molecule using oligonucleotide primersdesigned from this fragment. One primer was synthesized to initiate 5′extension of the known fragment, and the other primer was synthesized toinitiate 3′ extension of the known fragment The initial primers weredesigned using OLIGO 4.06 software (National Biosciences), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 mmol of each primer, reaction buffer containing Mg²⁺,(NH₂)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (AmershamBiosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Biosciences). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) tofill-in restriction site overhangs, and transfected into competent E.coli cells. Transformed cells were selected on antibiotic-containingmedia, and individual colonies were picked and cultured overnight at 37°C. in 384-well plates in LB/2× carb liquid media.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene)with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3,and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C.DNA was quantified by PICOGREEN reagent (Molecular Probes) as describedabove. Samples with low DNA recoveries were reamplified using the sameconditions as described above. Samples were diluted with 20%dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energytransfer sequencing primers and the DYENAMIC DIRECT kit (AmershamBiosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the aboveprocedure or are used to obtain 5′regulatory sequences using the aboveprocedure along with oligonucleotides designed for such extension, andan appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in SECP EncodingPolynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms(SNPs) were identified in SEQ ID NO:81-160 using the LIFESEQ database(Incyte Genomics). Sequences from the same gene were clustered togetherand assembled as described in Example III, allowing the identificationof all sequence variants in the gene. An algorithm consisting of aseries of filters was used to distinguish SNPs from other sequencevariants. Preliminary filters removed the majority of basecall errors byrequiring a minimum Phred quality score of 15, and removed sequencealignment errors and errors resulting from improper trimming of vectorsequences, chimeras, and splice variants. An automated procedure ofadvanced chromosome analysis analysed the original chromatogram files inthe vicinity of the putative SNP. Clone error filters used statisticallygenerated algorithms to identify errors introduced during laboratoryprocessing, such as those caused by reverse transcriptase, polymerase,or somatic mutation. Clustering error filters used statisticallygenerated algorithms to identify errors resulting from clustering ofclose homologs or pseudogenes, or due to contamination by non-humansequences. A final set of filters removed duplicates and SNPs found inimmunoglobulins or T-cell receptors.

Certain SNPs were selected for further characterization by massspectrometry using the high throughput MASSARRAY system (Sequenom, Inc.)to analyze allele frequencies at the SNP sites in four different humanpopulations. The Caucasian population comprised 92 individuals (46 male,46 female), including 83 from Utah, four French, three Venezualan, andtwo Amish individuals. The African population comprised 194 individuals(97 male, 97 female), all African Americans. The Hispanic populationcomprised 324 individuals (162 male, 162 female), all Mexican Hispanic.The Asian population comprised 126 individuals (64 male, 62 female) witha reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean,5% Vietnamese, and 8% other Asian. Allele frequencies were firstanalyzed in the Caucasian population; in some cases those SNPs whichshowed no allelic variance in this population were not further tested inthe other three populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:81-160 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Biosciences), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Biosciences). An aliquot containing 107counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1×saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualize using autoradiography or an alternative imaging meansandcompared.

XI. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting; see, e.g., Baldeschweiler et al., supra), mechanicalmicrospotting technologies, and derivatives thereof. The substrate ineach of the aforementioned technologies should be uniform and solid witha non-porous surface (Schena, M., ed. (1999) DNA Microarrays: APractical Approach, Oxford University Press, London). Suggestedsubstrates include silicon, silica, glass slides, glass chips, andsilicon wafers. Alternatively, a procedure analogous to a dot or slotblot may also be used to arrange and link elements to the surface of asubstrate using thermal, UV, chemical, or mechanical bonding procedures.A typical array may be produced using available methods and machineswell known to those of ordinary skill in the art and may contain anyappropriate number of elements (Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall,A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1Xfirst strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Biosciences). The reverse transcription reaction is performedin a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits(Incyte Genomics). Specific control poly(A)⁺ RNAs are synthesized by invitro transcription from noncoding yeast genomic DNA. After incubationat 37° C. for 2 hr, each reaction sample (one with Cy3 and another withCy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide andincubated for 20 minutes at 85° C. to the stop the reaction and degradethe RNA. Samples are purified using two successive CHROMA SPIN 30 gelfiltration spin columns (Clontech, Palo Alto Calif.) and aftercombining, both reaction samples are ethanol precipitated using 1 ml ofglycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.The sample is then dried to completion using a SpeedVAC (SavantInstruments Inc., Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2%SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Biosciences).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 μl ofarray element sample per slide.

Microarrays are V-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte Genomics). Array elements that exhibited atleast about a two-fold change in expression, a signal-to-backgroundratio of at least 2.5, and an element spot size of at least 40% wereidentified as differentially expressed.

Expression

The effects upon liver metabolism and hormone clearance mechanisms areimportant to understand the pharmacodynamics of a drug. For example, thehuman C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cellline, isolated from a 15-year-old male with liver tumor), which wasselected for strong contact inhibition of growth. The use of a clonalpopulation enhances the reproducibility of results obtained using thecells. C3A cells have many characteristics of primary human hepatocytesin culture: i) expression of insulin receptor and insulin-like growthfactor II receptor; ii) secretion of a high ratio of serum albumincompared with α-fetoprotein iii) conversion of ammonia to urea andglutamine; iv) ability to metabolize aromatic amino acids; and v)proliferation in glucose-free and insulin-free medium. The C3A cell lineis well established as an in vitro model of the mature human liver(Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997)Am J Physiol 272:G408-G416). SEQ ID NO:96 showed differential expressionin C3A cells treated with a variety of steroids includingbeclomethasone, medroxyprogesterone, budesonide, prednisone,dexamethasone, and progesterone, versus untreated C3A cells, asdetermined by microarray analysis. Specifically, the expression of SEQID NO:96 was increased at least 2-fold by treatment of cells with: 1-100microM medroxyprogesterone for 1-6 hours, 1-100 microM budesonide for1-6 hours, 1-100 microM progesterone for 1 hour, and 1-100 microMbetamethasone Therefore, SEQ ID NO:96 is useful for the diagnosis andmonitoring of liver, endocrine, and reproductive diseases and in thediagnosis of and as a therapeutic target for inflammatory diseases andhumoral immune response.

In an alternative example, the gene expression profile of nonmalignantprimary mammary epithelial cells (HMECs) was compared to that of variousbreast carcinoma lines at different stages of tumor progression. Thebreast carcinoma lines studied were BT-20, MCF7, MDA-mb-435S, Sk-BR-3,and T-47D. SEQ ID NO: 115 was found to be downregulated by at leasttwo-fold in MCF7, Sk-BR-3, and T-47D. Therefore SEQ ID NO: 115, encodingSEQ ID NO:35 can be used in assays to detect breast cancer.

In an alternative example, SEQ ID NO: 115 was downregulated by at leasttwo-fold in osteosarcoma tissues when compared to its expression innormal osteoblast primary culture cells, the NHO_(st)5488 cells, in fourout of the seven donors studied. Therefore, SEQ ID NO: 115, encoding SEQID NO:35 can be used in assays to detect osteosarcoma.

In an alternative example, human preadipocytes were treated with humaninsulin and PPAR-γ agonist for 3 days and subsequently were switched tomedium containing insulin for 24 hours, 48 hours, 4 days, 1.1 week, and2.1 weeks before the cells were collected for analysis. Differentiatedadipocytes were compared to untreated preadipocytes maintained inculture in the absence of inducing agents. SEQ ID NO: 115 wasdownregulated by at least two-fold in the differentiated adipocytesafter a minimum of 48 hours in the medium containing insulin andremained so for a maximum of 1.1 week. Therefore, through learning thegene expression profile during adipogenesis in humans, it will bepossible to understand the fundamental mechanism of adiposityregulation. Furthermore, by comparing the gene expression profiles ofadipogenesis in normal weight and donor with obesity it will be possibleto identify crucial genes, which might be potential drug targets forobesity and type II diabetes. SEQ ID NO:115, encoding SEQ ID NO:35 canbe used in the above assays.

In an alternative example, SEQ ID NO: 125 showed differential expressionin colon tissue from patients with colon cancer compared to matchedmicroscopically normal tissue from the same donors as determined bymicroarray analysis. The expression of SECP-45 was increased at leasttwo-fold in cancerous colon tissue. SEQ ID NO: 125 also showeddifferential expression in prostate LNCaP carcinoma cells compared toprostate PrEC epithelial cells as determined by microarray analysis. TheLNCaP cell line was isolated from a lymph node biopsy of a 50-year oldmale with metastatic prostate carcinoma. The expression of SECP-45 wasdecreased at least two-fold in prostate LNCaP carcinoma cells comparedto prostate PrEC epithelial cells. In an alternative example, SEQ ID NO:125 showed differential expression associated with immune andinflammatory responses as determined by microarray analysis. Theexpression of SEQ ID NO: 125 was increased by at least two-fold inperipheral blood mononuclear cells (PBMCs; 12% B lymphocytes, 40% Tlymphocytes, 20% NK cells, 25% monocytes, and 3% various cells thatinclude dendritic and progenitor cells) treated with interleukin-1β(IL-1β), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α)compared to untreated PBMCs; IL-1β is a cytokine that plays roles inacute inflammatory responses, fever induction, metabolic regulation, andbone remodeling. IL-1β induces its own production in monocytes and alsoinduces production of adhesion molecules and chemokines in endothelialcells and interferon-γ in NK cells. IL-6 plays roles in host defense,immune responses, and hematopoiesis. TNF-α is a pleiotropic cytokineinvolved in immune regulation and inflammatory responses. SEQ ID NO: 125also showed at least 2-fold decreased expression in human T cellleukemia Jurkat cells treated with a combination of the protein kinase Cactivator, phorbol myristate acetate (PMA), and the calcium ionophore,ionomycin, compared to untreated Jurkat cells as determined bymicroarray analysis. Treatment of T cells with PMA and ionomycin mimicsthe signaling events elicited during T cell activation. In addition, SEQID NO: 125 showed at least two-fold decreased expression in THP-1promonocyte cells stimulated with PMA and ionomycin. THP-1 is apromonocyte cell line isolated from the peripheral blood of a 1-year-oldmale with acute monocytic leukemia. THP-1 cells acquire monocyticcharacteristics in response to stimulation with PMA. Therefore, SEQ IDNO: 125 is useful in disease staging and diagnostic assays for cellproliferative disorders, including breast cancer, colon cancer, andprostate cancer, and autoimmune/inflammatory disorders.

In an alternative example, SEQ ID NO: 128 showed differential expressionin brain cingulate from a patient with Alzheimer's disease compared tomatched microscopically normal tissue from the same donor as determinedby microarray analysis. The expression of SECP-48 was increased at leasttwo-fold in cingulate tissue with Alzheimer's disease. Therefore, SEQ IDNO: 128 is useful in disease staging and diagnostic assays forneurological disorders, including Alzheimer's disease.

In an alternative example, human LNCaP is a prostate carcinoma cell lineisolated from a lymph node biopsy of a male donor with metastaticprostate carcinoma. LNCaP cells express prostate specific antigens andandrogen receptors, and produce prostatic acid phosphatase. PrEC is aprimary prostate epithelial cell line isolated from a normal donor. InLNCaP cells, one of three metastatic prostate carcinoma cell linestested, SEQ ID NO: 138 was downregulated at least two-fold when comparedwith PrEC cells.

In an alternative example, Jurkat is an acute T cell leukemia cell linethat grows actively in the absence of external stimuli. Jurkat has beenextensively used to study signaling in human T cells. PMA is a broadactivator of the protein kinase C-dependent pathways. Ionomycin is acalcium ionophore that permits the entry of calcium in the cell, henceincreasing the cytosolic calcium concentration. The combination of PMAand ionomycin activates two of the major signaling pathways used bymammalian cells to interact with their environment. In T cells, thecombination of PMA and ionomycin mimics the type of secondary signalingevents elicited during optimal B cell activation. SEQ ID NO: 149 wasdownregulated at least two-fold in the Jurkat T-cell leukemia cell linethat had been stimulated for one hour with 1 μM PMA (phorbol12-myristate 13-acetate) and with ionomycin concentrations varyingbetween 50 ng/ml and 1 μg/ml when compared to untreated Jurkat cells inthe absence of stimuli.

In an alternative example, TBP-1 is a promonocyte cell line that wasisolated from the peripheral blood of a 1-year-old male with acutemonocytic leukemia. Upon stimulation with PMA, THP-1 differentiates intoa macrophage-like cell that displays many characteristics of peripheralhuman macrophages. THP-1 cells have been extensively used in the studyof signaling in human monocytes and the identification of new factorsproduced by human monocytes. SEQ ID NO: 150 was downregulated at leasttwo-fold in THP-1 cells that had been stimulated for four or more hourswith 0.1 μM PMA and then further stimulated with 1 μg/ml ionomycin whencompared to untreated THP-1 cells in the absence of stimuli. Also, SEQID NO: 150 was upregulated at least two-fold in osteosarcoma tissue fromtwo donors with chondroblastic osteosarcoma of the femur when comparedwith a normal osteoblast cell line.

In an alternative example, a pure human mammary epithelial cell (HMEC)population was compared to breast carcinoma lines at various stages oftumor progression. SEQ ID NO: 156 was found to be downregulated at leasttwo fold in BT-20, BT-474, BT-483, Hs578T, MCF7, MDA-MB-468. ThereforeSEQ ID NO: 156 can be used in assays to detect breast cancer.

In an alternative example, the aim was to identify genes differentiallyregulated during the process of tumor progression. To this end, the geneexpression profiles of primary prostate epithelial cells and prostatecarcinomas that are representative of the different stages of tumorprogression were compared. SEQ ID NO: 156 was found to be downregulatedat least two fold in DU 145, LNCaP, and PC-3. Therefore, SEQ ID NO: 156can be used in assays to detect prostrate cancer.

In an alternative example, SEQ ID NO: 157 showed differential expressionassociated with breast cancer, as determined by microarray analysis.Breast carcinoma cell lines at various stages of tumor progression werecompared to primary human breast epithelial cells. The breast carcinomacell lines include MCF7, a breast adenocarcinoma cell line derived fromthe pleural effusion of a 69-year-old female; T-47D, a breast carcinomacell line derived from a pleural effusion from a 54-year-old female withan infiltrating ductal carcinoma of the breast; Sk-BR-3, a breastadenocarcinoma cell line isolated from a malignant pleural effusion of a43-year-old female; BT-20, a breast adenocarcinoma isolated in vitrofrom cells emigrating out of thin slices of a tumor mass isolated from a74-year-old female; MDA-mb-231, a breast tumor cell line isolated fromthe pleural effusion of a 51-year-old female, which forms poorlydifferentiated adenocarcinoma in nude mice and expresses the Wnt3oncogene, EGF and TGF-α; and MDA-mb-435S, a spindle shaped strain thatevolved from a cell line isolated from the pleural effusion of a 31 yearold female with metastatic, ductal adenocarcinoma of the breast. Thenonmalignant breast epithelial cell line, MCF-10A was isolated from a36-year-old woman with fibrocystic breast disease. All cell cultureswere propagated in a chemically-defined medium, according to thesupplier's recommendations and grown to 70-80% confluence prior to RNAisolation. The microarray experiments showed that expression of SEQ IDNO: 157 was up-regulated by at least two-fold in two of six cell linesexamined as compared to the nonmalignant breast epithelial cell line,MCF-10A.

In another experiment designed to investigate the process of tumorprogression and malignant transformation in breast tumors, a comparisonwas made against the primary mammary epithelial cell line HMEC, derivedfrom normal human mammary tissue (Clonetics, San Diego, Calif.) and thebreast tumor cell lines described above. The microarray experimentsindicated that expression of SEQ ID NO: 157 was decreased by at leasttwo fold in five breast tumor cells lines when compared to HMEC cells.Therefore, SEQ ID NO: 157 is useful in diagnostic and disease stagingassays for breast cancer and as a potential biological marker andtherapeutic agent in the treatment of breast cancer.

In an alternative example, SEQ ID NO: 157 also showed differentialexpression in prostate cancer, as determined by microarray analysis.Prostate carcinoma cell lines at various stages of tumor progressionwere compared to pr prostate epithelial cells. The prostate carcinomacell lines include: DU145, a prostate carcinoma cell line with nodetectable sensitivity to hormones, isolated from a metastatic site inthe brain of a 69-year-old male, that does not express prostate specificantigen; LNCaP, a prostate carcinoma cell line that expresses androgenreceptors and prostate specific antigen and was isolated from a lymphnode of a 50-year-old male with metastatic prostate cancer; and PC3, aprostate adenocarcinoma cell line isolated from a metastatic site in thebone of a 62-year-old male with grade IV prostate adenocarcinoma. Theprimary prostate epithelial cells, PrECs, were isolated from a normaldonor. All cell cultures were propagated in a chemically-defined medium,according to the supplier's recommendations and grown to 70-80%confluence prior to RNA isolation. The microarray experiments showedthat expression of SEQ ID NO: 157 was decreased by at least two-fold inall three prostate carcinoma cell lines, as well as in prostatemetastatic samples from brain, bone and nodes, as compared to primaryprostate epithelial cells. Therefore, SEQ ID NO: 157 is useful indiagnostic and staging assays for prostate cancer and as a potentialbiological marker and therapeutic agent in the treatment of prostatecancer.

In an alternative example, SEQ ID NO: 158 showed differential expressionin breast carcinoma cell lines versus primary mammary epithelial cellsas determined by microarray analysis. The breast carcinoma cell linesinclude BT20, a breast carcinoma cell line derived in vitro from cellsemigrating out of thin slices of a tumor mass isolated from a74-year-old female; BT474, a breast ductal carcinoma cell line isolatedfrom a solid, invasive ductal carcinoma of the breast from a 60-year-oldfemale; BT483, a breast ductal carcinoma cell line isolated from apapillary invasive ductal tumor from a 23-year-old normal, menstruating,parous female; HS578T, a breast ductal carcinoma cell line isolated froma 74-year-old female with breast carcinoma; MCF7, a breastadenocarcinoma cell line derived from the pleural effusion of a69-year-old female; and MDA-mb-468, a breast adenocarcinoma cell lineisolated from the pleural effusion of a 51-year-old female withmetastatic adenocarcinoma of the breast. The primary mammary epithelialcell line HMEC was derived from normal human mammary tissue (Clonetics,San Diego, Calif.). The microarray experiments showed that theexpression of SEQ ID NO: 158 were decreased by at least four fold in allsix breast carcinoma lines (BT20, DT474, BT483, HS578T, MCF7, andMDA-mb468) relative to cells from the primary mammary epithelial cellline, HMEC. Therefore, SEQ ID NO: 158 is useful as a diagnostic markeror as a potential therapeutic target for breast cancer.

In an alternative example, SEQ ID NO: 158 also showed differentialexpression in prostate carcinoma cell lines versus normal prostateepithelial cells as determined by microarray analysis. Three prostatecarcinoma cell lines, DU 145, LNCaP, and PC-3 were included in theexperiments. DU 145 was isolated from a metastatic site in the brain ofa 69 year old male with widespread metastatic prostate carcinoma. DU 145has no detectable sensitivity to hormones; forms colonies in semi-solidmedium; is only weekly positive for acid phosphatase; and cells arenegative for prostate specific antigen (PSA). LNCaP is a prostatecarcinoma cell line isolated from a lymph node biopsy of a 50 year oldmale with metastatic prostate carcinoma. LNCaP expresses PSA, producesprostate acid phosphatase, and expresses androgen receptors. PC-3, aprostate adenocarcinoma cell line, was isolated from a metastatic sitein the bone of a 62 year old male with grade IV prostate adenocarcinoma.The normal epithelial cell line, PrEC, is a primary prostate epithelialcell line isolated from a normal donor. In one experiment, theexpression of cDNAs from the prostate carcinoma cell lines were comparedto that of the normal prostate epithelial cells grown under the sameconditions (in the absence of growth factors and hormones). Thisexperiment showed that the expression of SEQ ID NO: 158 was decreased byat least four fold in both all three prostate carcinoma lines relativeto PrECs. In the other experiment, the expression of cDNAs from theprostate carcinoma cell lines grown in optimal conditions (in thepresence of growth factors and hormones) were compared to that of thenormal prostate epithelial cells grown under restrictive conditions (inthe absence of growth factors and hormones). The experiment showed thatthe expression of SEQ ID NO: 158 was also decreased by at least fourfold in DU145, LNCaP, and PC-3 prostate carcinoma lines relative toPrECs. Therefore, SEQ ID NO: 158 is useful as a diagnostic marker or asa potential therapeutic target for prostate cancers.

XII. Complementary Polynucleotides

Sequences complementary to the SECP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring SECP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of SECP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the SECP-encoding transcript.

XIII. Expression of SECP

Expression and purification of SECP is achieved using bacterial orvirus-based expression systems. For expression of SECP in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express SECP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof SECP in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding SECP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945).

In most expression systems, SECP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamBiosciences). Following purification, the GST moiety can beproteolytically cleaved from SECP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel et al.(supra, ch. 10 and 16). Purified SECP obtained by these methods can beused directly in the assays shown in Examples XVII, XVIII, and XIX whereapplicable.

XIV. Functional Assays

SECP function is assessed by expressing the sequences encoding SECP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1plasmid (Invitrogen), both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, for example, an endothelial or hematopoietic cellline, using either liposome formulations or electroporation. 1-2 μg ofan additional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GPP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyaridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York N.Y.).

The influence of SECP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding SECPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding SECP and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XV. Production of SECP Specific Antibodies

SECP substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizeanimals (e.g., rabbits, mice, etc.) and to produce antibodies usingstandard protocols.

Alternatively, the SECP amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skin in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art (Ausubel et al.,supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity (Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide and anti-SECP activity by, forexample, binding the peptide or SECP to a substrate, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring SECP Using Specific Antibodies

Naturally occurring or recombinant SECP is substantially purified byimmunoaffinity chromatography using antibodies specific for SECP. Animmunoaffinity column is constructed by covalently coupling anti-SECPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Biosciences). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing SECP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof SECP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/SECP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and SECPis collected.

XVII. Identification of Molecules Which Interact with SECP

SECP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133:529-539). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled SECP, washed, and anywells with labeled SECP complex are assayed. Data obtained usingdifferent concentrations of SECP are used to calculate values for thenumber, affinity, and association of SECP with the candidate molecules.

Alternatively, molecules interacting with SECP are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989;Nature 340:245-246), or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech).

SECP may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVIII. Demonstration of SECP Activity

An assay for growth stimulating or inhibiting activity of SECP measuresthe amount of DNA synthesis in Swiss mouse 3T3 cells (McKay, L and I.Leigh, eds. (1993) Growth Factors: A Practical Approach, OxfordUniversity Press, New York, N.Y.). In this assay, varying amounts ofSECP are added to quiescent 3T3 cultured cells in the presence of[³H]thymidine, a radioactive DNA precursor. SECP for this assay can beobtained by recombinant means or from biochemical preparations.Incorporation of [³H]thymidine into acid-precipitable DNA is measuredover an appropriate time interval, and the amount incorporated isdirectly proportional to the amount of newly synthesized DNA. A lineardose-response curve over at least a hundred-fold SECP concentrationrange is indicative of growth modulating activity. One unit of activityper milliliter is defined as the concentration of SECP producing a 50%response level, where 100% represents maximal incorporation of[³H]thymidine into acid-precipitable DNA.

Alternatively, an assay for SECP activity measures the stimulation orinhibition of neurotransmission in cultured cells. Cultured CHOfibroblasts are exposed to SECP. Following endocytic uptake of SECP, thecells are washed with fresh culture medium, and a whole cellvoltage-clamped Xenopus myocyte is manipulated into contact with one ofthe fibroblasts in SECP-free medium. Membrane currents are recorded fromthe myocyte. Increased or decreased current relative to control valuesare indicative of neuromodulatory effects of SECP (Morimoto, T. et al.(1995) Neuron 15:689-696).

Alternatively, an assay for SECP activity measures the amount of SECP insecretory, membrane-bound organelles. Transfected cells as describedabove are harvested and lysed. The lysate is fractionated using methodsknown to those of skill in the art, for example, sucrose gradientultracentrifugation. Such methods allow the isolation of subcellularcomponents such as the Golgi apparatus, ER, small membrane-boundvesicles, and other secretory organelles. Immunoprecipitations fromfractionated and total cell lysates are performed using SECP-specificantibodies, and immunoprecipitated samples are analyzed using SDS-PAGEand immunoblotting techniques. The concentration of SECP in secretoryorganelles relative to SECP in total cell lysate is proportional to theamount of SECP in transit through the secretory pathway.

Alternatively, AMP binding activity is measured by combining SECP with³²P-labeled AMP. The reaction is incubated at 37° C. and terminated byaddition of trichloroacetic acid. The acid extract is neutralized andsubjected to gel electrophoresis to remove unbound label. Theradioactivity retained in the gel is proportional to SECP activity.

XIX. Demonstration of Immunoglobulin Activity

An assay for SECP activity measures the ability of SECP to recognize andprecipitate antigens from serum. This activity can be measured by thequantitative precipitin reaction. (Golub, E. S. et al. (1987)Immunology: A Synthesis, Sinauer Associates, Sunderland, Mass., pp.113-115.) SECP is isotopically labeled using methods known in the art.Various serum concentrations are added to constant amounts of labeledSECP. SECP-antigen complexes precipitate out of solution and arecollected by centrifugation. The amount of precipitable SECP-antigencomplex is proportional to the amount of radioisotope detected in theprecipitate. The amount of precipitable SECP-antigen complex is plottedagainst the serum concentration. For various serum concentrations, acharacteristic precipitin curve is obtained, in which the amount ofprecipitable SECP-antigen complex initially increases proportionatelywith increasing serum concentration, peaks at the equivalence point, andthen decreases proportionately with further increases in serumconcentration. Thus, the amount of precipitable SECP-antigen complex isa measure of SECP activity which is characterized by sensitivity to bothlimiting and excess quantities of antigen.

Alternatively, an assay for SECP activity measures the expression ofSECP on the cell surface. cDNA encoding SECP is transfected into anon-leukocytic cell line. Cell surface proteins are labeled with biotin(de la Fuente, M. A. et al. (1997) Blood 90:2398-2405).Immunoprecipitations are performed using SECP-specific antibodies, andimmunoprecipitated samples are analyzed using SDS-PAGE andimmunoblotting techniques. The ratio of labeled immunoprecipitant tounlabeled immunoprecipitant is proportional to the amount of SECPexpressed on the cell surface.

Alternatively, an assay for SECP activity measures the amount of cellaggregation induced by overexpression of SECP. In this assay, culturedcells such as NIH3T3 are transfected with cDNA encoding SECP containedwithin a suitable mammalian expression vector under control of a strongpromoter. Cotransfection with cDNA encoding a fluorescent markerprotein, such as Green Fluorescent Protein (CLONTECH), is useful foridentifying stable transfectants. The amount of cell agglutination, orclumping, associated with transfected cells is compared with thatassociated with untransfected cells. The amount of cell agglutination isa direct measure of SECP activity.

Various modifications and variations of the described compositions,methods, and systems of the invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.It will be appreciated that the invention provides novel and usefulproteins, and their encoding polynucleotides, which can be used in thedrug discovery process, as well as methods for using these compositionsfor the detection, diagnosis, and treatment of diseases and conditions.Although the invention has been described in connection with certainembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Nor shouldthe description of such embodiments be considered exhaustive or limitthe invention to the precise forms disclosed. Furthermore, elements fromone embodiment can be readily recombined with elements from one or moreother embodiments. Such combinations can form a number of embodimentswithin the scope of the invention. It is intended that the scope of theinvention be defined by the following claims and their equivalents.TABLE 1 Incyte Incyte Polypeptide Incyte Polynucleotide PolynucleotideProject ID SEQ ID NO: Polypeptide ID SEQ ID NO: ID Incyte Full LengthClones 1417062 1 1417062CD1 81 1417062CB1 90110408CA2, 90110416CA2,90110432CA2, 90172377CA2, 90172385CA2, 90172453CA2, 90172461CA2,90172477CA2, 90172485CA2 2007701 2 2007701CD1 82 2007701CB1 2915695 32915695CD1 83 2915695CB1 2969449 4 2969449CD1 84 2969449CB1 2994102 52994102CD1 85 2994102CB1 90110580CA2, 90110588CA2, 90110596CA2 3410251 63410251CD1 86 3410251CB1 90126170CA2, 90126194CA2, 90126262CA2 5330327 75330327CD1 87 5330327CB1 5532048 8 5532048CD1 88 5532048CB1 56002716  956002716CD1 89 56002716CB1 56002716CA2, 90110503CA2, 90110511CA2,90110535CA2, 90110619CA2 60129797  10 60129797CD1 90 60129797CB190109831CA2, 90109847CA2 6246243 11 6246243CD1 91 6246243CB190188842CA2, 90188850CA2, 90188874CA2, 90188890CA2, 90188902CA2,90188982CA2 6804755 12 6804755CD1 92 6804755CB1 6804755CA2, 90125796CA26856852 13 6856852CD1 93 6856852CB1 6856852CA2, 90166714CA2,90166730CA2, 90166738CA2, 90166746CA2, 90166814CA2, 90166822CA2,90166830CA2, 90166838CA2 7482027 14 7482027CD1 94 7482027CB1 7493507 157493507CD1 95 7493507CB1 3075994 16 3075994CD1 96 3075994CB190164903CA2, 90164911CA2, 90164927CA2, 90165019CA2, 90165027CA2,90165043CA2 2378119 17 2378119CD1 97 2378119CB1 1483648CA2, 2075676CA2,2378119CA2, 90166558CA2, 90166582CA2, 90166690CA2, 90166757CA2 298741818 2987418CD1 98 2987418CB1 2987418CA2, 5476092CA2, 90166703CA2,90166803CA2, 90166827CA2 4223862 19 4223862CD1 99 4223862CB1 1597349CA2,4223862CA2, 90166968CA2, 90166992CA2, 90167185CA2, 90167277CA2 604640620 6046406CD1 100 6046406CB1 6046406CA2, 90166102CA2, 90166118CA2,90166134CA2, 90166218CA2, 90166226CA2 6743529 21 6743529CD1 1016743529CB1 2474053CA2, 6743529CA2, 90166357CA2 7283809 22 7283809CD1 1027283809CB1 7283809CA2 7637563 23 7637563CD1 103 7637563CB1 7637563CA27663814 24 7663814CD1 104 7663814CB1 90166178CA2, 90166286CA2 8001939 258001939CD1 105 8001939CB1 90173305CA2, 90173313CA2, 90173337CA2,90173421CA2, 90173429CA2, 90173437CA2, 90173445CA2, 90188622CA2,90188654CA2, 90188662CA2, 90188670CA2, 90188678CA2, 90188686CA2,90188694CA2, 90188778CA2, 90188786CA2, 90188938CA2 8191019 26 8191019CD1106 8191019CB1 90166811CA2, 90166819CA2, 90166835CA2, 90167102CA2,90167118CA2, 90167126CA2, 90167134CA2, 90167142CA2, 90167202CA2,90167210CA2, 90167218CA2, 90167226CA2, 90188610CA2, 90188649CA2,90188657CA2, 90188665CA2, 90188673CA2, 90188681CA2, 90188689CA2,90188749CA2, 90188757CA2, 90188765CA2, 90188773CA2, 90188774CA2,90188781CA2, 90188789CA2, 90188809CA2, 90188941CA2  919788 27 919788CD1107 919788CB1 90177394CA2 4758058 28 4758058CD1 108 4758058CB1 749983529 7499835CD1 109 7499835CB1 90108956CA2, 90108988CA2, 90109132CA2,90109148CA2, 90109156CA2, 90109208CA2, 90109433CA2, 90110058CA2,90132951CA2, 90132967CA2, 90132975CA2, 90132991CA2, 90133051CA2,90133059CA2, 90133067CA2, 90133075CA2, 90133091CA2, 90133161CA2,90133193CA2, 90133709CA2, 90133725CA2, 90133733CA2, 90133741CA2,90133817CA2, 90133825CA2, 90133833CA2, 90135904CA2, 90135912CA2,90135944CA2, 90136020CA2 2484647 30 2484647CD1 110 2484647CB11726009CA2, 1830692CA2, 6574257CA2, 8612535CA2, 90132931CA2,90132955CA2, 90132963CA2, 90132971CA2, 90133008CA2, 90133039CA2,90133055CA2, 90133063CA2, 90133087CA2, 90133103CA2, 90133123CA2,90133127CA2, 90133215CA2, 90133219CA2, 90133224CA2, 90133235CA2,90133251CA2, 90133311CA2 2587034 31 2587034CD1 111 2587034CB1 2702991 322702991CD1 112 2702991CB1 2702991CA2 2744736 33 2744736CD1 1132744736CB1 2744736CA2, 90133729CA2, 90133745CA2, 90133845CA2 2915475 342915475CD1 114 2915475CB1 2915475CA2, 90132915CA2, 90133015CA2,90133022CA2, 90133023CA2, 90133031CA2, 90133150CA2 3040427 35 3040427CD1115 3040427CB1 1824963CA2 7499722 36 7499722CD1 116 7499722CB1 677690937 6776909CD1 117 6776909CB1 7985313CA2, 90132902CA2, 90132910CA2,90132918CA2, 90133002CA2, 90133010CA2, 90133018CA2, 90133034CA2,90133044CA2, 90133953CA2 7280438 38 7280438CD1 118 7280438CB17280438CA2, 8018238CA2, 90133544CA2, 90133644CA2 7499809 39 7499809CD1119 7499809CB1 7499921 40 7499921CD1 120 7499921CB1 2705858 412705858CD1 121 2705858CB1 55115172CA2, 90109710CA2, 90109766CA2,90109774CA2, 90109866CA2, 90109902CA2, 90109909CA2, 90109917CA2,90109925CA2, 90109933CA2, 90109941CA2, 90109949CA2, 90109957CA2,90109965CA2, 90109973CA2, 90109981CA2, 90109989CA2, 90110002CA2,90110025CA2, 90110041CA2, 90110057CA2, 90110065CA2, 90110073CA2,90110081CA2, 90110089CA2, 90110450CA2, 90175707CA2, 90175715CA2,90175739CA2, 90175807CA2, 90175823CA2, 90175831CA2, 90175847CA2 306989242 3069892CD1 122 3069892CB1 90160650CA2, 90160666CA2, 90160690CA2,90160758CA2, 90160766CA2, 90160818CA2, 90160826CA2, 90160834CA2,90160902CA2, 90160910CA2, 90160918CA2, 90160926CA2, 90160934CA2,90160942CA2, 90187851CA2, 90187867CA2, 90187891CA2, 90187951CA2,90187967CA2, 90187991CA2, 90188268CA2, 90188276CA2, 90188284CA2,90188292CA2, 90188360CA2, 90188368CA2, 90188384CA2, 90188428CA2,90188451CA2, 90188459CA2, 90188465CA2, 90188467CA2, 90188475CA2,90188483CA2, 90188491CA2, 90188551CA2, 90188559CA2, 90188567CA2,90188575CA2, 90188583CA2, 90188591CA2 3069586 43 3069586CD1 1233069586CB1 90125322CA2 7500104 44 7500104CD1 124 7500104CB1 7500203 457500203CD1 125 7500203CB1 4843802 46 4843802CD1 126 4843802CB14843802CA2, 90110741CA2, 90110817CA2, 90110825CA2, 90110833CA2,90172510CA2, 90172518CA2, 90172526CA2, 90172634CA2, 90172642CA2 587752247 5877522CD1 127 5877522CB1 5877522CA2, 6120870CA2, 90110153CA2,90110161CA2, 90110177CA2, 90110193CA2, 90110253CA2, 90110261CA2,90110269CA2, 90110277CA2  617491 48 617491CD1 128 617491CB1 90109750CA2,90109758CA2, 90109782CA2, 90109850CA2, 90109858CA2, 90109874CA2 628990149 6289901CD1 129 6289901CB1 90115210CA2, 90115234CA2 6817709 506817709CD1 130 6817709CB1 7272661CA2 6849312 51 6849312CD1 1316849312CB1 90190001CA2, 90190009CA2, 90190025CA2, 90190033CA2,90190041CA2, 90190109CA2, 90190117CA2, 90190125CA2 7409581 52 7409581CD1132 7409581CB1 7437113 53 7437113CD1 133 7437113CB1 90155830CA2 750026054 7500260CD1 134 7500260CB1 90025555CA2, 90025563CA2, 90025587CA2,90025588CA2, 90025595CA2, 90025663CA2, 90025671CA2 7659504 55 7659504CD1135 7659504CB1  821165 56 821165CD1 136 821165CB1 821165CA2,90109761CA2, 90109769CA2, 90109853CA2, 90109861CA2, 90109869CA2,90109877CA2 7499672 57 7499672CD1 137 7499672CB1 90110796CA2,90111017CA2, 90111033CA2 7500276 58 7500276CD1 138 7500276CB11218389CA2, 1875737CA2, 8168187CA2 1440723 59 1440723CD1 139 1440723CB190110787CA2, 90172328CA2, 90172336CA2, 90172368CA2, 90172444CA2 747961260 7479612CD1 140 7479612CB1 90133152CA2, 90133176CA2, 90133184CA2,90133192CA2, 90133252CA2, 90133260CA2, 90133268CA2, 90133284CA2,90133292CA2, 90134004CA2 1391514 61 1391514CD1 141 1391514CB1 7292618CA22102578 62 2102578CD1 142 2102578CB1 90132950CA2, 90132958CA2,90132966CA2, 90132982CA2, 90132990CA2, 90133058CA2, 90133066CA2,90133082CA2, 90133090CA2, 90133331CA2, 90197343CA2 3213122 63 3213122CD1143 3213122CB1 3213122CA2, 6322461CA2, 90133962CA2, 90133986CA2,90134054CA2, 90134062CA2, 90134070CA2, 90134078CA2, 90172690CA2 432630764 4326307CD1 144 4326307CB1 6037749 65 6037749CD1 145 6037749CB16037749CA2 6285519 66 6285519CD1 146 6285519CB1 7131125CA2, 7317881CA270336045  67 70336045CD1 147 70336045CB1 7625761CA2, 90144712CA2 715357768 7153577CD1 148 7153577CB1 7153577CA2, 90132905CA2, 90132961CA2,90132977CA2, 90132985CA2, 90133053CA2, 90133061CA2, 90133069CA2,90133077CA2, 90133093CA2, 90133129CA2, 90133205CA2, 90133361CA2,90133601CA2, 90133633CA2, 90133641CA2 7500299 69 7500299CD1 1497500299CB1 7480218 70 7480218CD1 150 7480218CB1 7501159 71 7501159CD1151 7501159CB1 90189746CA2 7501932 72 7501932CD1 152 7501932CB1 750111173 7501111CD1 153 7501111CB1 90023351CA2, 90023363CA2 7501113 747501113CD1 154 7501113CB1 7501118 75 7501118CD1 155 7501118CB190023315CA2 7501128 76 7501128CD1 156 7501128CB1 90023351CA2,90023363CA2, 90023391CA2 7501920 77 7501920CD1 157 7501920CB1 7510325 787510325CD1 158 7510325CB1 90012168CA2, 90012176CA2, 90012240CA2,90012276CA2, 90012412CA2, 90023320CA2, 90023354CA2, 90023359CA2,90023361CA2, 90023362CA2, 90023367CA2, 90023370CA2, 90023377CA2,90023383CA2, 90176711CA2, 90176803CA2, 90176827CA2, 90176835CA2,90176843CA2, 90177559CA2, 90177691CA2 7510966 79 7510966CD1 1597510966CB1 90023363CA2, 90023395CA2, 90177567CA2 7386101 80 7386101CD1160 7386101CB1

TABLE 2 Polypeptide Incyte GenBank ID NO: SEQ Polypeptide or PROTEOMEProbability ID NO: ID ID NO: Score Annotation 5 2994102CD1 g13536533.0E−07 [fl][Strongylocentrotus purpuratus] sperm receptor for egg jelly(Moy, G. W. et al. (1996) J. Cell Biol. 133 (4), 809-817) 15 7493507CD1g1046223 1.1E−185 [Homo sapiens] melanoma ubiquitous mutated protein(Coulie, P. G. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92 (17),7976-7980) 16 3075994CD1 g3747097 9.9E−91 [Homo sapiens] C1q-relatedfactor 25 8001939CD1 g19850565 2.0E−55 [fl][Mus musculus] NFATactivation molecule 1 28 4758058CD1 g205250 5.0E−18 [Rattus norvegicus]Ly6C antigen (Friedman, S. et al. (1990) Immunogenetics 31 (2), 104-111)29 7499835CD1 g285971 8.5E−58 [Homo sapiens] PAP homologous protein(Itoh, T. et al. (1993) Biochim. Biophys. Acta 1172 (1-2), 184-186) 353040427CD1 g20799379 1.0E−66 [fl][Rattus norvegicus] neural stem cellderived neuronal survival protein precursor 43 3069586CD1 g142891836.7E−113 [Homo sapiens] chorein (Ueno, S. et al. (2001) Nat. Genet. 28(2), 121-122) 44 7500104CD1 g190484 2.3E−84 [Homo sapiens] preprosalivary proline-rich protein (Maeda, N. et al. (1985) J. Biol. Chem.260 (20), 11123-11130) 45 7500203CD1 g482909 8.5E−58 [Homo sapiens]pancreatitis-associated protein (Dusetti, N. J. et al. (1994) Genomics19 (1), 108-114) 57 7499672CD1 g13194528 1.5E−57 [Homo sapiens]NPC-related protein NAG73 58 7500276CD1 g794071 2.6E−56 [Macacafascicularis] epididymal secretory protein 14.6 (Perry, A. C. et al.(1995) Gene 153 (2), 291-292) 69 7500299CD1 g13543353 1.6E−68 [Homosapiens] (BC005839) follistatin-like 3 (secreted glycoprotein) 707480218CD1 g13241974 9.0E−289 [Homo sapiens] CocoaCrisp 71 7501159CD1g1747306 6.9E−156 [Mus musculus] SDR2 (Shirozu, M. et al. (1996)Genomics 37 (3), 273-280) 72 7501932CD1 g1488047 5.4E−14 [Xenopuslaevis] RING finger protein 73 7501111CD1 367644|Rn.25073 7.5E−16[Rattus norvegicus] [Receptor (signalling)] [Plasma membrane] G-protein-coupled receptor with a large extracellular domain, expressed in lung,kidney and heart Abe, J. et al. (1999) J. Biol. Chem. 274: 19957-19964Ig-hepta, a novel member of the G-protein-coupled hepta-helical receptor(GPCR) family that has immunoglobulin-like repeats in a long N- terminalextracellular domain and defines a new subfamily of GPCRs. 74 7501113CD1367644|Rn.25073 8.5E−20 [Rattus norvegicus] [Receptor (signalling)][Plasma membrane] G-protein- coupled receptor with a large extracellulardomain, expressed in lung, kidney and heart Abe, J. supra 76 7501128CD1367644|Rn.25073 2.5E−15 [Rattus norvegicus][Receptor(signalling)][Plasma membrane]G protein-coupled receptor with a largeextracellular domain, expressed in lung, kidney and heart 77 7501920CD1g5525078 5.0E−19 [Rattus norvegicus] seven transmembrane receptor (Abe,J. et al. (1999) J. Biol. Chem. 274 (28), 19957-19964) 77 7501920CD1367644|Rn.25073 4.3E−20 [Rattus norvegicus][Receptor(signalling)][Plasma membrane] G-protein-coupled receptor with a largeextracellular domain, expressed in lung, kidney and heart Abe, J. et al.Supra 80 7386101CD1 g458726 6.9E−27 [Homo sapiens] estrogen responsivefinger protein (efp) (Inoue, S. et al. (1993) Proc. Natl. Acad. Sci.U.S.A. 90 (23), 11117-11121)

TABLE 3 Amino Potential Potential Analytical SEQ Incyte Acid Phosphor-Glyco- Methods ID Polypeptide Resi- ylation sylation SignatureSequences, and NO: ID dues Sites Sites Domains and Motifs Databases 11417062CD1 269 S30 S68 S222 T204 Signal_cleavage: M1-G26 SPSCAN T248Signal Peptide: M25-A45, HMMER M25-A47, M25-G49 Non-cytosolic domains:M1-L269 TMHMMER Biotin represser PF01317: Q13-R29 BLIMPS_PFAM Leucinezipper pattern: L155-L176 MOTIFS Eukaryotic putative MOTIFS RNA-bindingregion RNP-1 signature: K74-F81 TonB-dependent receptor MOTIFS proteinssignature 1: M1- E98 2 2007701CD1 127 S108 S114 T3 T70 N79Signal_cleavage: M1-W33 SPSCAN T85 Signal Peptide: M11-N25, HMMERM11-A29, M11-W33, S10- W33, M11-A38, M1-Q30 Cytosolic domains: M1-M11,N67-W127 TMHMMER Transmembrane domains: L12-H34, L44-L66 TMHMMERNon-cytosolic domains: T35-L43 TMHMMER 3 2915695CD1 71 T64Signal_cleavage: M1-G21 SPSCAN Signal Peptide: M1-Q18, M1-G21, HMMERM1-S23, M1-G25 Non-cytosolic domains: M1-T71 TMHMMER 4 2969449CD1 83 S61Signal_cleavage: M1-A18 SPSCAN Signal Peptide: M1-A18 HMMERNon-cytosolic domains: M1-T83 TMHMMER Ribonucleotide reductase largePROFILESCAN subunit signature: A21-N72 5 2994102CD1 306 S58 S194 T17T162 N45 N52 N111 Signal_cleavage: M1-G60 SPSCAN T182 T255 T274Non-cytosolic domains: M1-R306 TMHMMER 6 3410251CD1 334 S70 S173 S224N147 Signal_cleavage: M1-A26 SPSCAN S321 Signal Peptide: P9-A26, M1-A26,HMMER M1-A30, L7-A26 Non-cytosolic domains: M1-V264 TMHMMERTransmembrane domains: V265-L287 TMHMMER Cytosolic domains: T288-A334TMHMMER Leucine Rich Repeat: A120-R143, HMMER_PFAM A144-P167, A96- G119,L168-P191, R72-G95 Leucine zipper pattern: L172-L193 MOTIFS 7 5330327CD1950 S6 S13 S37 S48 N644 N853 Signal_cleavage: M1-A35 SPSCAN S57 S68 S104S114 S141 S179 S186 S340 S344 S355 S388 S396 S411 S425 S441 S566 S607S611 S668 S680 S777 S779 S844 S855 S901 S916 T63 T110 T251 T262 T294T309 T333 T562 T679 T793 T824 T836 Non-cytosolic domains: M1-F950TMHMMER KINASE PIP5K BLAST_PRODOM TRANSFERASE PHOSPHATIDYLINOSITOL-4-PHOSPHATE FINGER-CONTAINING PHOSPHOINOSITIDE FYVE PTDINS4P-5- KINASE1- PHOSPHATIDYLINOSITOL- 4-PHOSPHATE 8 5532048CD1 546 S17 S37 S82 S87Signal_cleavage: M1-S58 SPSCAN S186 S199 S212 S221 S237 S296 S302 S375S402 S421 S439 S447 S488 T32 T412 T484 T493 T505 T517 T523 Non-cytosolicdomains: M1-I546 TMHMMER 9 56002716CD1 226 S72 S94 S108 S158Signal_cleavage: M1-A20 SPSCAN S205 T70 T118 Non-cytosolic domains:M1-I226 TMHMMER Signal Peptide: M1-T17, M1-A18, HMMER M1-A21, M1-V23,M1-A20 10 60129797CD1 130 S40 S108 T46 Signal_cleavage: M1-A23 SPSCANSignal Peptide: M1-V21, M1-A23, HMMER M1-S24, M1-G32, M1-T26Non-cytosolic domains: M1-S130 TMHMMER 11 6246243CD1 195 S4 S33 S40 S41N62 N159 Signal_cleavage: M1-L24 SPSCAN S114 S141 S146 S161 T65 T155Signal Peptide: M1-R25, M1-L24, M1-P26 HMMER Non-cytosolic domains:M1-Q195 TMHMMER 12 6804755CD1 112 S7 S42 Signal_cleavage: M1-A62 SPSCANSignal Peptide: M8-G27, M8-Q29, HMMER M8-A33, M1- A33, M8-P30 Cytosolicdomains: M1-G112 TMHMMER Aldo/keto reductase family signatures: M8-P73PROFILESCAN 13 6856852CD1 107 S22 S35 Signal_cleavage: M1-G16 SPSCANSignal Peptide: M1-G16, M1-A18, HMMER M1-S20, M1-S22, M1-R24, M1-S20Non-cytosolic domains: M1-L107 TMHMMER 14 7482027CD1 221 S100 T112 T155N91 Signal_cleavage: M1-G14 SPSCAN T211 Signal Peptide: M1-A15, HMMERM1-W19, M1-G20 Cytosolic domains: C210-Q221 TMHMMER Non-cytosolicdomains: M1-Q186 TMHMMER Transmembrane domains: A187-A209 TMHMMER 157493507CD1 642 S5 S22 S50 S61 N66 N142 N162 Signal_cleavage: M1-S37SPSCAN S78 S147 S165 S167 S240 S297 S306 S315 S318 S363 S372 S390 S393S439 S472 S527 S531 S601 S622 T44 T171 T270 T274 T545 T549 T579 T610Y615 Non-cytosolic domains: M1-R642 TMHMMER Cell attachment sequence:R72-D74 MOTIFS 16 3075994CD1 238 S15 S143 T224 Signal_cleavage: M1-S15SPSCAN Signal Peptide: M1-S15, HMMER M1-G18, M1-A20 Non-cytosolicdomains: M1-D238 TMHMMER C1q domain: A111-I235 HMMER_PFAM C1q domainproteins BL01113: G73-G99, V125- BLIMPS_BLOCKS V160, D194-K213,S228-P237 Complement C1Q domain BLIMPS_PRINTS signature PR00007: P119-K145, F146-G165, D194-D215, K226-Y236 PRECURSOR SIGNAL BLAST_PRODOMCOLLAGEN REPEAT HYDROXYLATION GLYCOPROTEIN CHAIN PLASMA EXTRACELLULARMATRIX PD002992: R118-I235 COLLAGEN ALPHA BLAST_PRODOM PRECURSOR CHAINREPEAT SIGNAL CONNECTIVE TISSUE EXTRACELLULAR MATRIX PD000007: G36-G99SIMILAR TO BLAST_PRODOM CUTICULAR COLLAGEN PD067228: G18-P102 PRECURSORSIGNAL BLAST_PRODOM COLLAGEN ALPHA 3IX CHAIN EXTRACELLULAR MATRIXCONNECTIVE TISSUE PD028299: G36-G98 C1Q DOMAIN DM00777 BLAST_DOMOP02746|70-250: G58-D238 BLAST_DOMO P23206|477-673: R63-P237 BLAST_DOMOS23297|465-674: P51-I234 BLAST_DOMO S49158|70-253: G58-D238 BLAST_DOMOC1q domain signature: F128-Y158 MOTIFS 17 2378119CD1 113 S49 S72 S90 T10signal_cleavage: M1-S49 SPSCAN T25 Y35 18 2987418CD1 97 S76 T68 SignalPeptide: M1-G23 HMMER signal_cleavage: M25-N92 SPSCAN inside: M1-T33TMHMMER TMhelix: F34-I56 TMHMMER outside: L57-K97 TMHMMER 19 4223862CD1147 S68 S80 signal_cleavage: M1-G36 SPSCAN inside: M1-H93 TMHMMERTMhelix: L94-L116 TMHMMER outside: G117-N147 TMHMMER 20 6046406CD1 95S45 S91 signal_cleavage: M1-A24 SPSCAN Signal Peptide: M1-A22 M1-A24HMMER 21 6743529CD1 76 N41 signal_cleavage: M1-G20 SPSCAN SignalPeptide: M1-C19 HMMER 22 7283809CD1 154 signal_cleavage: M1-S26 SPSCANSignal Peptide: M2-S19, M2-S24, HMMER M2-S26, M2-G31 23 7637563CD1 160S60 S79 T49 T61 signal_cleavage: M1-G24 SPSCAN T136 T140 Signal Peptide:M1-G24 HMMER 24 7663814CD1 72 S19 S50 N41 N55 signal_cleavage: M1-P21SPSCAN Signal Peptide: M1-P21, M1-P23, M1-S24 HMMER 25 8001939CD1 270S64 S92 S210 T36 N107 signal_cleavage: M1-G42 SPSCAN T97 T139 T199 T228T243 Signal Peptide: M1-G42 HMMER outside: M1-K163 TMHMMER TMhelix:L164-W186 TMHMMER inside: N187-L270 TMHMMER 26 8191019CD1 121signal_cleavage: M1-P18 SPSCAN Signal Peptide: M1-P18, M1-P21 HMMERF-actin capping protein beta PROFILESCAN subunit signature: G17- P92 27919788CD1 181 S87 signal_cleavage: M1-A41 SPSCAN 28 4758058CD1 120 S55signal_cleavage: M1-P22 SPSCAN Signal Peptide: M7-S21, HMMER M7-P24,M7-G26, M7-C29, M1-G26, M1-C29 u-PAR/Ly-6 domain: M1-V60, S83-L120HMMER_PFAM Ly-6/u-PAR domain proteins BLIMPS_BLOCKS BL00983: L12-L20,Q23-C32, A76-N91 LY-6/U-PAR DOMAIN BLAST_DOMO DM02129|P35460|1-133:BLAST_DOMO M1-L120 DM02129|I48639|1-134: BLAST_DOMO M1-L119DM02129|P09568|1-130: BLAST_DOMO M1-L120 DM02129|P05533|1-133:BLAST_DOMO M1-L120 29 7499835CD1 129 S35 S57 S60 S67 signal_cleavage:M1-G26 SPSCAN S77 T29 Signal Peptide: M13-T29, S9-G26, HMMER M5-G26, M5-T29, M1-E28, M1-G26, M5-V24 Lectin C-type domain: T29-F127 HMMER_PFAMC-type lectin domain BLIMPS_BLOCKS proteins BL00615: C51-E68, W112-C125C-type lectin domain PROFILESCAN signature and profile: D80- K128PRECURSOR SIGNAL BLAST_PRODOM PROTEIN LECTIN REG LITHOSTATHINEREGENERATING INFLAMMATORY RESPONSE ACUTE PD149843: L21-D65 C-TYPE LECTINBLAST_DOMO DM00035|Q06141|33-172: BLAST_DOMO G66-F127, L33-D65DM00035|P35230|33-172: BLAST_DOMO G66-F127, L33-D65DM00035|P23132|33-172: BLAST_DOMO D65-F127, L33-D65DM00035|S54979|33-171: BLAST_DOMO G66-F127, L33-D65 C-type lectin domainsignature: C100-C125 MOTIFS 30 2484647CD1 101 S82 signal_cleavage:M1-A36 SPSCAN Signal Peptide: M1-A19, M1-P21 HMMER 31 2587034CD1 83 T76signal_cleavage: M1-A28 SPSCAN Signal Peptide: M1-G21 HMMER Cytosolicdomain: I33-F83 TMHMMER Transmembrane domain: F10-L32 TMHMMERNon-cytosolic domain: M1-Y9 TMHMMER Pancreatic ribonuclease PROFILESCANfamily signature: N5-I61 Indole-3-glycerol phosphate PROFILESCANsynthase signature: L25- G77 Leucine zipper pattern: L11-L32 MOTIFS 322702991CD1 172 T14 signal_cleavage: M1-A35 SPSCAN 33 2744736CD1 168 S100S147 signal_cleavage: M1-A56 SPSCAN Signal Peptide: M24-P43 HMMER 342915475CD1 83 T37 signal_cleavage: M1-A18 SPSCAN Signal Peptide: M1-A18,M1-E20, HMMER M1-T22, M1-G26 Cytosolic domain: T72-T83 TMHMMERTransmembrane domain: L49-F71 TMHMMER Non-cytosolic domain: M1-H48TMHMMER 35 3040427CD1 167 S89 S134 T23 T66 signal_cleavage: M1-A47SPSCAN Signal Peptide: M24-A39, HMMER M24-C41, M24-G44, M24- A47,M22-E49, L28-A47, M22-A47 EF-hand clacium-binding BLMPS_BLOCKS domainprotein BL00018: D150-F162 Laminin-type EGF-like BLIMPS_BLOCKS (LE)domain protein BL01248: C1061-C1354 CALMODULIN REPEAT BLAST_DOMODM00011|JS0027|29-74: T116-A163 EF-HAND CALCIUM-BINDING DOMAINBLAST_DOMO DM00256|JS0027|1-27: M88-S115 Binding-protein-dependentMOTIFS transport systems inner membrane component. signature: M1-R29EF-hand calcium-binding domain: MOTIFS D102-L114, D150- F162 367499722CD1 195 S100 S161 S165 N123 N192 signal_cleavage: M1-A28 SPSCANS182 T76 T122 Signal Peptide: M1-G21 HMMER Cytosolic domain: I33-L195TMHMMER Transmembrane domain: F10-L32 TMHMMER Non-cytosolic domain:M1-Y9 TMHMMER Leucine zipper pattern: L11-L32 MOTIFS 37 6776909CD1 89S71 signal_cleavage: M1-G27 SPSCAN Signal Peptide: M1-G27, M1-A29 HMMER38 7280438CD1 136 S51 S115 signal_cleavage: M1-A16 SPSCAN SignalPeptide: M1-A16, HMMER M1-G18, M1-G20 39 7499809CD1 420 S90 S180 S274N210 signal_cleavage: M1-C18 SPSCAN S383 S413 T46 T319 T336 T353 T388Signal Peptide: M1-C18, M1-V20, HMMER M1-G23, M1-G27 Regulator ofchromosome MOTIFS condensation (RCC1) signature 2: V140-L150 Leucinezipper pattern: L153-L174, MOTIFS L160-L181, L167- L188, L303-L324 407499921CD1 667 S68 S144 S186 N659 signal_cleavage: M1-G46 SPSCAN S229S230 S231 S245 S339 S471 S475 S494 S627 S644 T89 T102 T221 T411 T603Flavodoxin: G78-V126 HMMER_PFAM Cytosolic domain: M1-I19 TMHMMERTransmembrane domain: N20-I42 TMHMMER Non-cytosolic domain: K43-C667TMHMMER 41 2705858CD1 83 signal_cleavage: M1-S18 SPSCAN Signal Peptide:M1-S18, M1-T20, HMMER M1-A23, M1-R25 42 3069892CD1 80 S7 S46 S47 S62signal_cleavage: M1-A24 SPSCAN T72 Signal Peptide: M1-A24, M20-A51 HMMER43 3069586CD1 367 S54 S62 S82 S87 signal_cleavage: M1-P29 SPSCAN S326S332 T27 T36 T110 T112 PROTEIN VACUOLAR BLAST_PRODOM SORTINGASSOCIATEDVPS13 TIPC T08G11.1 PD025730: V2-E360 44 7500104CD1 154 S15 S47signal_cleavage: M1-A16 SPSCAN Signal Peptide: M1-A16, M1-D18 HMMERSALIVARY ACIDIC PROLINE RICH BLAST_PRODOM PHOSPHOPROTEIN 1/2 PRECURSORPRP1/PRP 3 PRP2/PRP4 PIFF/PIFS PROTEIN A/PROTEIN C CONTAINS: PEPTIDE PCREPEAT SALIVA SIGNAL PAROTID GLAND PHOSPHORYLATION PD054888: M1-D55PROTEIN REPEAT BLAST_PRODOM SIGNAL PRECURSOR PRION GLYCOPROTEIN NUCLEARGPI ANCHOR BRAIN MAJOR PD001091: R34-Q154 COLLAGEN ALPHA BLAST_PRODOMPRECURSOR CHAIN REPEAT SIGNAL CONNECTIVE TISSUE EXTRACELLULAR MATRIXPD000007: G66- P153 TRACHEAL COLONIZATION BLAST_PRODOM FACTOR PRECURSORSIGNAL SARCAL UMENIN CALCIUM BINDING GLYCOPROTEIN ALTERNATIVE SPLICINGPD136752: D18-G150 PROLINE-RICH PROTEIN BLAST_DOMODM01369|P02810|86-164: BLAST_DOMO P74-P153 DM01281|P04280|17-124:BLAST_DOMO Q42-G141 DM03894|A39066|1-159: BLAST_DOMO M1-Q154DM01281|P04280|212-315: BLAST_DOMO G56-G146 45 7500203CD1 129 S35 S57S60 S67 signal_cleavage: M1-G26 SPSCAN S77 T29 Signal Peptide: M13-T29,HMMER M5-G26, M5-T29, M1- E28, M1-G26, M5-V24 Lectin C-type domain:T29-F127 HMMER_PFAM C-type lectin domain proteins BLIMPS_BLOCKS BL00615:C51-E68, W112-C125 C-type lectin domain signature PROFILESCAN andprofile: D80-K128 PRECURSOR SIGNAL PROTEIN LECTIN REG BLAST_PRODOMLITHOSTATHINE REGENERATING INFLAMMATORY RESPONSE ACUTE PD149843: L21-D65C-TYPE LECTIN BLAST_DOMO DM00035|Q06141|33-172: BLAST_DOMO G66-F127,L33-D65 DM00035|P35230|33-172: BLAST_DOMO G66-F127, L33-D65DM00035|P23132|33-172: BLAST_DOMO D65-F127, L33-D65DM00035|S54979|33-171: BLAST_DOMO G66-F127, L33-D65 C-type lectin domainsignature: C100-C125 MOTIFS 46 4843802CD1 116 S65 S66 S72 T89signal_cleavage: M1-G23 SPSCAN Signal Peptide: M1-G23, M1-S30, HMMERM1-A25, M1-G28 47 5877522CD1 84 T14 signal_cleavage: M1-R16 SPSCANSignal Peptide: M1-R16 HMMER 48 617491CD1 83 S26 S40 T39 N37signal_cleavage: M1-S28 SPSCAN Signal Peptide: M1-A23, M1-S30, M1-S28HMMER 49 6289901CD1 133 S40 S72 S89 S97 signal_cleavage: M1-G38 SPSCANMyelin proteolipid protein PROFILESCAN signatures: D10-A61 50 6817709CD1117 S52 T28 T30 signal_cleavage: M1-A15 SPSCAN Signal Peptide: M1-A15HMMER 51 6849312CD1 99 S23 S80 T69 N19 signal_cleavage: M1-S23 SPSCANSignal Peptide: M1-S23 HMMER 52 7409581CD1 114 S38 S58 S61 T35signal_cleavage: M1-G34 SPSCAN Signal Peptide: M1-G34 HMMER Cytosolicdomain: Q33-G114 TMHMMER Transmembrane domain: P15-S32 TMHMMERNon-cytosolic domain: M1-Q14 TMHMMER 53 7437113CD1 699 S99 S101 S152 N97N333 N352 signal_cleavage: M1-S33 SPSCAN S236 S280 S284 N490 N524 N613S312 S327 S335 S441 S459 S492 S501 S502 S590 S631 S636 T54 T67 T294 T298T375 T398 T399 T403 T475 T545 T547 T604 T675 T684 54 7500260CD1 144 S115N53 signal_cleavage: M1-A51 SPSCAN 55 7659504CD1 382 S17 S109 S195 N154N263 signal_cleavage: M1-G35 SPSCAN S279 T105 T139 T175 T227 Cellattachment sequence: R32-D34 MOTIFS Leucine zipper pattern: L329-L350MOTIFS 56 821165CD1 93 signal_cleavage: M1-Q46 SPSCAN Signal Peptide:M1-A17, M1-P19, HMMER M1-S24, M1-L25, M1-P18, M1-S23 57 7499672CD1 110S65 signal_cleavage: M1-C61 SPSCAN 58 7500276CD1 115 S33 S85 N99signal_cleavage: M1-A19 SPSCAN Signal Peptide: M1-A16, M1-A19, HMMERM1-P21, M1-Q23, M1-F24 E1 family: A6-I111 HMMER_PFAM PRECURSOR SIGNALALLERGEN PROTEIN BLAST_PRODOM MITE SECRETORY E1 POLYMORPHISM DER IIPD008264: S31-I111, M1-C27 59 1440723CD1 161 S133 signal_cleavage:M1-G47 SPSCAN Signal Peptide: M1-V21 HMMER 60 7479612CD1 88 T38signal_cleavage: M1-T38 SPSCAN Signal Peptide: M1-V15 HMMER 611391514CD1 79 S18 S75 signal_cleavage: M1-C23 SPSCAN Signal Peptide:M1-C23 HMMER 62 2102578CD1 76 S18 S25 T63 signal_cleavage: M1-A24 SPSCANSignal Peptide: M1-A24 HMMER 63 3213122CD1 116 S29 S67 signal_cleavage:M1-Q15 SPSCAN Cytochrome c family heme- MOTIFS binding site signature:C106-S111 64 4326307CD1 558 S21 S82 S128 S158 N86 N262 N327signal_cleavage: M1-S21 SPSCAN S237 S242 S244 S296 S300 S318 S329 S339S365 S386 S484 S543 T135 T226 T346 T448 T449 T476 Y49 Y143 SignalPeptide: M1-S21 HMMER 65 6037749CD1 155 T36 T77 T109 N67 N98 N122signal_cleavage: M1-A25 SPSCAN T124 T143 Y151 Signal Peptide: M7-S23,M1-A25, M7-A25 HMMER 66 6285519CD1 77 T7 signal_cleavage: M1-C32 SPSCANSignal Peptide: L14-C32, M8-G31, M8-C32 HMMER 67 70336045CD1 240 S11 S56S76 S90 N202 signal_cleavage: M1-A44 SPSCAN S149 S178 S192 S204 S205T238 EF hand: D96-L124, G132-A160 HMMER_PFAM 68 7153577CD1 101 S32 S36S47 S52 signal_cleavage: M1-A33 SPSCAN Signal Peptide: M1-A20, M1-S26HMMER 69 7500299CD1 129 S121 T112 N81 signal_cleavage: M1-S26 SPSCANSignal Peptide: M1-S26, M1-A20 HMMER Kazal-type serine proteaseHMMER_PFAM inhibitor domain: C66- C109 Osteonectin domain signatures:Q46-C88 PROFILESCAN KAZAL PROTEINASE INHIBITOR BLAST_DOMODM00123|P50291|186- 238: S55-C109 70 7480218CD1 500 S64 S81 S98 S136 N28signal_cleavage: M1-A20 SPSCAN S176 S207 S245 S278 S279 S367 T4 T30 T58T273 T398 T471 Y249 SCP-like extracellular HMMER_PFAM protein: Q63-G214Extracellular proteins BLIMPS_BLOCKS SCP/Tpx-1/Ag5/PR-1/Sc7 proteinsBL01009: M86-C103, H133-Y146, T166- C186, V200-H215 Allergen V5/Tpx-1family BLIMPS_PRINTS signature PR00837: C165- C181, Y201-G214, M86-L104,H133-Y146 Venom allergen 5 signature BLIMPS_PRINTS PR00838: M86-L104,T131-Y146, V164-I183 PROTEIN PRECURSOR BLAST_PRODOM SIGNALPATHOGENESISRELATED ANTIGEN ALLERGEN VENOM MULTIGENE FAMILY AG5PD000542: P78-G214 FSG 120K CYSRICH BLAST_PRODOM PROTEIN GLYCOPROTEINEGFLIKE DOMAIN PD128352: G53-G232 EXTRACELLULAR BLAST_DOMO PROTEINSSCP/TPX- 1/AG5/PR-1/SC7 DM00332|P48060|1-175: I57-Y218 EXTRACELLULARBLAST_DOMO PROTEINS SCP/TPX- 1/AG5/PR-1/SC7 DM00332|P54108|11-182: T58-W212 EXTRACELLULAR BLAST_DOMO PROTEINS SCP/TPX- 1/AG5/PR-1/SC7DM00332|P16562|9-180: Q63- N211 EXTRACELLULAR BLAST_DOMO PROTEINSSCP/TPX- 1/AG5/PR-1/SC7 DM00332|P35778|12-207: T58- P217 Extracellularproteins MOTIFS SCP/Tpx-1/Ag5/PR-1/Sc7 signature 2: Y201-W212 717501159CD1 402 S116 S121 S194 N138 N309 N322 signal_cleavage: M1-N22SPSCAN S305 S358 T57 T95 T106 T145 T172 T225 T276 T289 Y129 SignalPeptide: M1-V20 HMMER Reeler domain: S31-K156 HMMER_PFAM SDR2 PROTEINPD139571: Q164-S367 BLAST_PRODOM PROTEIN SDR2 BASIC BLAST_PRODOMHEMOLYMPH PRECURSOR SIGNAL PD035283: P24-S163 72 7501932CD1 363 S27 S193S293 N178 signal_cleavage: M1-S31 SPSCAN SPRY domain: A227-P349HMMER_PFAM PF00622 Domain in Spla BLIMPS_PFAM and the RyanodineReceptors (SPRY domain) PROTEIN FINGER MIDLINE BLAST_PRODOM ZINC FINGERRING STONUSTOXIN PUTATIVE TRANSCRIPTION FACTOR XPRF PD002421: K171-L343RFP TRANSFORMING PROTEIN BLAST_DOMO DM01944|I49642|513-634: G228-C346RFP TRANSFORMING PROTEIN BLAST_DOMO DM01944|A49656|508-630: G228-C346 737501111CD1 221 S115 T13 T30 T55 N139 N168 Signal_cleavage: M1-G17 SPSCANT104 T155 Signal Peptide: M1-G17, M1-G19, HMMER M1-G20, M1-G23 747501113CD1 267 S115 S219 S225 N139 N168 N205 Signal_cleavage: M1-G17SPSCAN S248 T13 T30 T55 T104 T155 Signal Peptide: M1-G17, M1-G19, HMMERM1-G20, M1-G23 75 7501118CD1 236 S115 S225 T13 T30 N139 N168signal_cleavage: M1-G17 SPSCAN T55 T104 T155 Signal Peptide: M1-G17,M1-G19, HMMER M1-G20, M1-G23 76 7501128CD1 221 S115 T13 T30 T55 N139N168 signal_cleavage: M1-G17 SPSCAN T104 T155 Signal Peptide: M1-G17,M1-G19, HMMER M1-G20, M1-G23 PHD-finger. PF00628: C105-P119 BLIMPS_PFAM77 7501920CD1 410 S96 S200 S206 N120 N149 N186 signal_cleavage: M1-G17SPSCAN S229 S247 S283 N263 N291 N298 S350 S357 S401 N310 N335 N349 T13T36 T85 T136 T265 T351 Signal Peptide: M1-G17, HMMER M1-G19, M1-G20,M1-G23 78 7510325CD1 67 signal_cleavage: M1-P50 SPSCAN Sigma-54interaction domain PROFILESCAN signatures and profile: L9-R56 Eukaryoticmitochondrial PROFILESCAN porin signature: M3-F66 79 7510966CD1 49 807386101CD1 495 S166 S181 S325 N310 SPRY domain: A359-P481 HMMER_PFAMS425 T118 T153 Zinc finger, C3HC4 type HMMER_PFAM (RING finger): C12-C50Zinc finger, C3HC4 type PROFILESCAN (RING finger), signature: L6- R61Zinc finger C3HC4 type BLIMPS_BLOCKS BL00518: C27-C35 PROTEIN FINGERMIDLINE BLAST_PRODOM ZINCFINGER RING STONUSTOXIN PUTATIVE TRANSCRIPTIONFACTOR XPRF PD002421: V316-L475 RFP TRANSFORMING PROTEIN DM01944BLAST_DOMO I49642|513-634: G360-C478 A49656|508-630: G360-C478 ZINCFINGER, C3HC4 BLAST_DOMO TYPE, DM00063 A49656|6-55: L6-R51A43906|137-187: E8-E52 Zinc finger, C3HC4 type MOTIFS (RING finger),signature: C27-I36

TABLE 4 Polynucleotide SEQ ID NO:/ Incyte ID/ Sequence Length SequenceFragments 81/1417062CB1/ 1-233, 1-528, 1-1146, 10-618, 17-281, 17-567,20-285, 20-664, 23-316, 23-524, 25-874, 28-335, 127-578, 184-853; 1146742-1145, 786-984, 786-1016, 796-1063 82/2007701CB1/ 1-81, 18-489,18-702, 187-702, 315-702, 363-656, 364-702, 491-663, 581-702, 589-10431043 83/2915695CB1/ 1-704, 31-667, 31-733, 31-955, 32-1684, 44-578,59-689, 60-629, 73-785, 74-785, 97-415, 191-447, 209-1192, 210- 1684936, 227-497, 272-936, 274-1180, 280-785, 392-646, 492-524, 492-539,492-542, 492-548, 492-554, 492-559, 492-569, 492-572, 492-584, 492-599,492-614, 499-785, 522-600, 522-644, 529-644, 552-638, 552-644, 559-644,582-644, 589-644, 612-644, 619-644, 709-964, 711-964, 715-964, 725-962,821-1120, 836-1122, 1269-1373, 1269- 1377, 1271-1366, 1313-1377,1369-1422 84/2969449CB1/ 1-755, 39-642, 47-857, 76-871, 217-922,224-748, 228-736, 240-978, 252-633, 252-922, 252-977, 280-758, 283- 1584995, 317-839, 319-839, 321-772, 321-967, 322-767, 327-799, 330-790,330-814, 335-820, 342-820, 351-894, 402-975, 406-939, 408-944, 431-980,462-1062, 471-1060, 547-980, 556-1410, 560-1217, 560-1272, 566-1235,572- 1079, 580-1115, 598-1297, 625-1584, 657-1584, 666-1311, 670-1580,687-1340, 717-1583, 790-1584, 810-1270, 857-1584, 869-1584, 918-1329,924-1584, 1074-1362, 1074-1584, 1097-1584, 1109-1584, 1121-1356,1135-1584, 1141-1584, 1142-1584, 1185-1584, 1274-1583 85/2994102CB1/1-274, 1-1490, 19-684, 27-326, 27-434, 38-607, 39-709, 49-357, 641-884,641-1199, 846-1114, 863-1474, 866- 1490 1089, 866-1090, 914-989,990-1490, 1372-1476 86/3410251CB1/ 1-233, 1-754, 310-494, 310-797,382-635, 429-814, 646-1073, 655-1108, 667-1074, 673-1096, 692-1073,700-1102, 1418 703-1073, 717-1073, 766-1093, 770-1048, 771-1073,790-1189, 790-1418, 864-1033 87/5330327CB1/ 1-296, 1-467, 75-710,75-717, 75-792, 334-868, 399-922, 456-790, 805-1511, 819-2112, 821-1335,839-1335, 845- 3485 1081, 857-1345, 858-1299, 868-1345, 933-1345,1095-1335, 1138-1409, 1339-1878, 1343-1857, 1343-1973, 1346-1734,1346-1751, 1346-1786, 1346-1794, 1346-1867, 1346-1868, 1346-1881,1346-1882, 1346-1898, 1346- 1912, 1346-1933, 1348-1944, 1424-1976,1444-1948, 1550-2410, 1571-1938, 1571-2161, 1573-2173, 1701-2410,1763-2141, 1772-2153, 1779-2410, 1806-2410, 1842-2410, 1854-2488,1861-2410, 1875-2410, 1877-2112, 1880- 2410, 1973-2591, 1991-2410,2111-2488, 2165-2410, 2168-2410, 2504-2739, 2504-2763, 2504-2974,2571-3415, 2641-3372, 2665-2927, 2781-2931, 2859-3485 88/5532048CB1/1-772, 1-834, 1-3044, 13-220, 108-780, 382-599, 416-693, 416-878,416-1064, 434-870, 434-953, 491-758, 594- 3427 1129, 602-1084, 619-985,619-1529, 638-942, 642-1096, 705-1243, 718-912, 779-1028, 783-1087,788-1376, 836- 1657, 955-1176, 976-1560, 976-1601, 981-1295, 983-1215,983-1380, 983-1554, 986-1248, 986-1437, 1069-1210, 1101- 1622,1101-1797, 1101-1941, 1102-1663, 1103-1659, 1189-1673, 1200-1923,1242-1856, 1251-1990, 1260-1726, 1271-1473, 1284-1924, 1292-1823,1299-1503, 1324-1580, 1335-2003, 1336-1959, 1353-1891, 1369-2003, 1387-1512, 1395-1988, 1457-1950, 1480-1763, 1499-1584, 1541-2081, 1541-2083,1549-1758, 1609-1888, 1623-2003, 1652-1958, 1652-1994, 1654-2332,1656-2004, 1658-2327, 1739-1999, 1773-1943, 1825-2081, 1926-2093, 1946-1987, 2036-2451, 2061-2451, 2238-2661, 2376-2451, 2395-2448, 2530-3044,2691-3033, 2753-3427, 2820-3427, 2863-3427, 2909-3427, 2964-3216,2964-3402 89/56002716CB1/ 1-27, 1-398, 1-457, 1-495, 1-496, 1-575,1-664, 1-767, 1-780, 1-811, 5-766, 15-670, 34-885, 45-860, 56-669, 210-1438 911, 576-1357, 663-1434, 786-1333, 840-1356, 849-1356, 886-1438,1413-1434 90/60129797CB1/ 1-453, 2-1710, 27-454, 111-566, 134-382,163-411, 279-1035, 444-1008, 471-1152, 543-1121, 572-1334, 603-1334,1710 657-1334, 698-907, 708-1274, 725-983, 725-996, 725-1232, 749-997,751-959, 756-1030, 756-1320, 764-1050, 769-1055, 793-1047, 811-972,812-1116, 819-1066, 829-1112, 829-1114, 829-1306, 829-1353, 829-1366,829-1368, 830-1407, 831-1473, 833-1395, 834-1507, 836-1356, 837-1352,839-1101, 843-1667, 851-1169, 859-1128, 861-1092, 862-1122, 871-1460,872-1079, 876-1449 91/6246243CB1/ 1-100, 1-640, 3-577, 7-381, 154-628,154-655, 181-753, 236-753, 272-736, 275-732, 341-731, 342-652, 347-734,753 368-732, 404-734, 411-734, 645-736 92/6804755CB1/ 1-220, 1-258,1-369, 1-420, 1-499, 1-513, 1-526, 1-649, 3-634, 7-627, 32-844, 87-420,202-692, 256-723, 295-1137, 1780 312-892, 379-1033, 409-918, 436-1103,481-1212, 510-1091, 551-1100, 630-1419, 658-1019, 680-1183, 693-1097,699-782, 699-794, 699-798, 699-802, 699-945, 700-761, 700-1200, 703-759,703-761, 703-790, 703-794, 703-802, 703-803, 703-839, 703-848, 703-989,703-1014, 703-1033, 703-1039, 704-792, 705-839, 705-1014, 706-802, 707-780, 707-802, 707-902, 713-945, 716-765, 716-789, 716-902, 716-931,716-945, 716-947, 716-989, 717-800, 717- 945, 717-975, 717-977, 717-987,717-989, 718-787, 718-945, 719-802, 719-935, 722-802, 722-975, 723-934,730- 792, 730-802, 731-979, 734-1021, 738-802, 747-878, 747-887,747-1058, 747-1065, 747-1077, 750-1077, 757-989, 760-989, 760-1033,761-1033, 763-902, 763-1192, 770-1192, 778-1215, 784-1033, 786-1077,810-900, 810-1005, 810-1077, 811-1005, 811-1077, 816-945, 816-1023,820-989, 820-1059, 833-1077, 834-1172, 839-1077, 850-979, 858-943,858-979, 858-989, 858-992, 858-1065, 858-1077, 860-902, 860-1077,861-989, 869-945, 869-1077, 872- 950, 876-1054, 881-1033, 892-945,892-987, 892-1077, 904-987, 904-1067, 906-985, 912-1062, 912-1077,916-987, 919-1077, 924-1719, 927-1023, 927-1031, 931-1031, 935-1009,935-1010, 935-1031, 935-1075, 938-1010, 945- 1077, 946-1052, 946-1065,946-1077, 955-1026, 958-1037, 974-1077, 975-1049, 975-1057, 975-1063,975-1065, 975-1075, 976-1064, 999-1077, 1002-1160, 1002-1405, 1003-1075,1010-1562, 1032-1077, 1034-1077, 1191-1701, 1331-1401, 1336-178093/6856852CB1/ 1-573, 1-580, 15-577 580 94/7482027CB1/ 1-394, 66-495,70-726, 292-731, 300-730, 337-729, 419-729 731 95/7493507CB1/ 1-806,10-504, 10-2758, 24-648, 29-200, 29-606, 32-324, 127-421, 204-705,579-1233, 657-1327, 662-1313, 684- 2758 1219, 726-1402, 732-1351,739-1369, 741-1402, 767-1419, 780-1099, 833-1494, 836-1384, 850-1351,851-1406, 855-1457, 874-1521, 875-1418, 910-1506, 934-1486, 963-1606,995-1430, 1013-1621, 1059-1456, 1084-1745, 1102- 1309, 1110-1382,1111-1785, 1158-1761, 1202-1771, 1208-1323, 1212-1735, 1227-1787,1229-1451, 1229-1474, 1251-1874, 1251-1909, 1283-1876, 1294-1552,1325-1795, 1326-2043, 1331-1830, 1347-1726, 1350-2000, 1362- 1627,1373-2077, 1392-1705, 1425-1475, 1427-1686, 1427-1982, 1433-2054,1444-1693, 1450-1998, 1470-1733, 1470-1739, 1470-1935, 1471-1774,1510-2288, 1512-2129, 1520-1795, 1530-2141, 1541-1832, 1546-2257, 1550-2191, 1554-2315, 1568-1847, 1579-1835, 1580-2130, 1582-2110, 1616-2308,1617-1862, 1617-2165, 1627-2238, 1633-2152, 1647-1918, 1648-1913,1677-2301, 1703-2250, 1703-2339, 1709-2293, 1730-2248, 1734-2347, 1737-1985, 1762-2086, 1766-2298, 1773-2034, 1777-2018, 1783-2602, 1785-2352,1808-2320, 1815-2488, 1843-2443, 1870-2096, 1870-2347, 1893-2250,1895-2203, 1943-2135, 1943-2191, 1943-2249, 1943-2501, 1962-2212,1962-2239, 1972-2239, 1977-2561, 1982-2649, 2005-2529, 2012-2645,2020-2486, 2039-2278, 2039-2293, 2039- 2602, 2063-2700, 2066-2696,2068-2664, 2082-2707, 2087-2339, 2121-2491, 2127-2745, 2131-2758,2160-2712, 2170-2717, 2170-2723, 2171-2712, 2174-2722, 2181-2712,2182-2712, 2211-2721, 2222-2712, 2234-2551, 2240- 2712, 2255-2721,2267-2640, 2271-2721, 2275-2716, 2278-2679, 2283-2714, 2285-2538,2288-2706, 2288-2721, 2296-2722, 2305-2593, 2320-2721, 2328-2722,2340-2721, 2351-2721, 2360-2718, 2373-2636, 2385-2499, 2388-2544, 2425-2715, 2439-2719, 2468-2715, 2471-2716, 2471-2718, 2499-2758, 2526-2718,2535-2670, 2535-2718, 2587-2709, 2587-2721, 2651-2719 96/3075994CB1/1-1361, 490-531, 550-816, 555-1189, 659-1298, 757-1043, 778-1295,815-1361, 933-1383 1383 97/2378119CB1/ 1-230, 28-560, 46-343, 49-288,50-298, 50-561, 51-342, 52-557, 53-224, 56-317, 56-338, 56-545, 59-552,59-558, 826 59-564, 59-566, 69-382, 69-559, 70-191, 70-392, 71-361,72-513, 72-522, 72-526, 74-297, 74-338, 76-522, 76-565, 77-354, 77-355,78-222, 78-351, 78-377, 80-342, 80-343, 80-356, 80-364, 82-347, 83-340,83-539, 83-576, 84-409, 85-550, 87-569, 89-336, 89-562, 91-362, 92-549,93-378, 93-445, 93-466, 96-382, 97-383, 102-546, 106-345, 106-383, 106-560, 109-368, 110-358, 110-362, 110-549, 111-305, 111-329, 111-358,111-377, 111-378, 111-402, 111-403, 111-560, 111-561, 111-565, 112-355,113-349, 113-610, 114-553, 115-375, 116-526, 117-561, 120-380, 128-546,128-549, 129-547, 132-550, 132-568, 139-401, 144-552, 151-550, 154-550,169-533, 182-551, 203-506, 214-546, 225-552, 253-374, 253-561, 254-491,275-525, 290-826, 309-491, 332-560, 332-608, 344-560, 360-55498/2987418CB1/ 1-279, 1-700, 1-1025, 107-959, 422-570, 422-856,473-1006, 620-1025, 732-1025, 782-1025 1025 99/4223862CB1/ 1-893,380-593, 380-907, 398-908, 398-1221, 404-691, 404-704, 404-865, 404-927,537-1223, 669-1195, 794-908, 1223 930-978, 1063-1111 100/6046406CB1/1-548, 1-549, 34-549 549 101/6743529CB1/ 1-229, 3-520, 42-520, 52-157,85-520, 89-520 520 102/7283809CB1/ 1-347, 1-480, 1-485, 1-501, 1-541,1-746, 1-926, 91-950, 347-927, 383-950, 394-927, 413-930, 427-927,428-927, 950 438-927, 471-949 103/7637563CB1/ 1-520, 1-589, 1-630,172-913 913 104/7663814CB1/ 1-640, 57-561 640 105/8001939CB1/ 1-587,1-617, 18-612, 18-617, 175-393, 452-1082, 505-1113, 666-716, 824-11131113 106/8191019CB1/ 1-440, 2-442, 3-442, 294-615, 294-762, 294-811,295-811, 297-769, 345-811, 372-933, 457-811, 480-811, 514-811 933107/919788CB1/ 1-638, 13-937, 44-639, 82-662, 222-792, 236-790, 239-793,240-792, 288-787, 290-792, 333-640, 362-792, 374- 1280 627, 383-659,387-792, 394-781, 401-662, 409-793, 418-694, 419-675, 451-615, 451-617,451-619, 451-646, 451- 651, 451-661, 451-705, 451-718, 451-719, 451-734,451-742, 451-749, 451-753, 451-773, 451-785, 451-792, 451-812, 451-833,451-841, 451-861, 451-868, 451-874, 451-1029, 451-1048, 452-623,452-640, 454-712, 466-1176, 467-743, 473-793, 490-793, 494-938, 499-792,513-1145, 516-773, 516-791, 520-1280, 529-712, 532-1212, 547- 719,568-1015, 589-682, 589-793, 589-1010, 704-894 108/4758058CB1/ 1-555,1-682, 37-555, 365-527, 365-534, 384-521, 384-522, 393-697 697109/7499835CB1/ 1-174, 20-263, 27-279, 28-285, 28-294, 30-239, 30-259,30-294, 31-243, 34-275, 36-251, 36-270, 36-273, 37-226, 723 37-253,37-254, 52-581, 63-249, 63-279, 71-289, 71-293, 73-291, 74-292, 75-264,75-268, 75-294, 81-289, 87-294, 88-260, 89-294, 90-264, 90-276, 90-294,91-294, 92-285, 95-268, 96-251, 214-702, 257-472, 285-723, 295-699, 295-706, 295-720, 303-525, 313-550, 315-723, 317-546, 317-549, 325-565,333-514, 341-609, 347-703, 350-545, 363-565, 363-630, 364-580, 364-583,364-589, 364-592, 365-658, 372-590, 376-655, 381-608, 410-717, 422-717,427-649, 427-658, 427-664, 457-634, 474-630, 475-691 110/2484647CB1/1-663, 8-551, 8-1049, 93-673, 189-907, 262-618, 267-748, 285-543,302-687, 302-705, 302-748, 302-815, 307-787, 1049 322-798, 323-592,323-721, 324-581, 324-593, 324-642, 324-655, 324-688, 329-882, 338-809,354-900, 365-614, 373-750, 375-748, 376-652, 377-635, 396-593, 400-795,437-702, 453-907, 454-898, 475-751, 476-764, 483-907, 492-781, 499-642,529-907, 535-824, 542-648, 558-1041, 566-798, 567-814, 580-845, 587-845,587-907, 588-815, 598-845, 605-905, 612-748, 618-905, 636-907, 645-748,650-730, 665-882, 671-748 111/2587034CB1/ 1-360, 97-220, 128-333 360112/2702991CB1/ 1-246, 1-432, 1-450, 1-507, 1-522, 1-523, 1-527, 1-553,1-570, 1-578, 1-583, 1-609, 1-618, 1-621, 1-660, 1-673, 1- 1466 684,1-688, 1-696, 1-725, 1-726, 1-818, 1-856, 149-624, 152-961, 192-692,230-475, 230-510, 235-745, 253-726, 259-483, 261-591, 288-662, 328-526,335-1081, 412-1081, 423-658, 425-1006, 428-869, 459-1081, 479-1083,508-1073, 663- 1236, 663-1240, 663-1270, 670-1270, 714-1279, 734-1244,739-1282, 825-1302, 846-1413, 851-1393, 868-1424, 871-1466, 873-1210,898-1427, 905-1192, 906-1461, 919-1192, 942-1203, 958-1202, 968-1253,976-1237, 1018- 1439, 1029-1296, 1034-1309, 1047-1343, 1058-1273,1060-1307, 1112-1150, 1112-1151, 1114-1285, 1148-1187 113/2744736CB1/1-233, 1-253, 1-384, 1-388, 1-467, 1-477, 1-487, 1-538, 1-592, 1-594,1-596, 1-645, 1-650, 1-791, 12-844, 118-828, 1724 197-433, 210-658,214-658, 222-813, 222-963, 231-712, 341-517, 359-671, 444-949, 446-931,474-1034, 479-948, 496-887, 496-974, 503-918, 509-1243, 511-1156,536-1147, 539-854, 548-1023, 556-956, 558-944, 606-1407, 629-1198, 649-784, 653-927, 704-1338, 715-1128, 755-1060, 755-1188, 756-1354,776-1393, 794-953, 805-1388, 821-1231, 857-1338, 882-1479, 887-1724,892-1393, 949-1418, 974-1414, 986-1207, 996-1435, 1184-1311, 1255-1397114/2915475CB1/ 1-284, 1-528, 1-579, 1-631, 1-740, 1-748, 1-760, 1-761,1-778, 3-473, 3-550, 3-756, 3-758, 3-761, 60-538, 252-778, 778 254-774,635-778, 701-778 115/3040427CB1/ 1-271, 1-484, 1-511, 1-521, 1-532,1-590, 19-617, 34-663, 86-721, 116-600, 265-716, 333-891, 362-634,363-897, 1974 366-1033, 370-1052, 374-1008, 376-1066, 379-1037, 383-614,385-683, 385-972, 412-645, 412-674, 412-677, 412- 843, 412-981, 412-985,414-518, 416-890, 423-1046, 446-821, 465-637, 470-770, 471-679, 473-696,474-1052, 483- 868, 489-821, 489-905, 497-706, 497-744, 498-947,498-1036, 500-950, 501-686, 501-763, 519-870, 526-902, 527- 1047,531-970, 557-1052, 565-962, 567-962, 572-1036, 574-1075, 581-1034,588-962, 589-638, 589-905, 592-1036, 593-1052, 594-1030, 594-1056,605-1045, 606-845, 614-1036, 616-1047, 625-1036, 626-1036, 632-1052,639-905, 642-1036, 651-1052, 652-1052, 656-905, 657-905, 668-1030,691-1013, 693-1052, 697-850, 709-1044, 712-1052, 723-1052, 730-1052,734-1030, 762-1036, 766-1052, 770-1030, 770-1052, 772-1052, 783-1050,786-1036, 805- 1052, 809-1052, 835-1052, 836-1052, 843-1116, 857-1052,858-1052, 858-1178, 860-1052, 860-1218, 860-1448, 872-1052, 872-1158,877-1052, 883-1052, 883-1146, 889-1045, 907-1403, 913-1384, 957-1027,1080-1605, 1404- 1974, 1706-1734, 1738-1766 116/7499722CB1/ 1-334,1-990, 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1247-2158, 1251-2158, 1253-2158, 1254-2161, 1255-2158,1255-2230, 1256-2103, 1256-2158, 1257-2049, 1257-2158, 1260-2158,1265-2158, 1270-2175, 1273-2158, 1275-2158, 1276- 2171, 1278-2158,1279-1672, 1280-2158, 1281-2158, 1283-2158, 1284-2158, 1287-2158,1288-2067, 1288-2110, 1290-2138, 1295-2156, 1302-2084, 1302-2158,1311-2158, 1313-2158, 1314-2158, 1315-2158, 1318-2134, 1321-2252,1336-1848, 1340-1530, 1342-1849, 1347-2158, 1348-1998, 1349-2080,1353-2158, 1354-2158, 1356- 2188, 1357-2158, 1367-1849, 1367-1997,1373-1849, 1382-2158, 1385-2158, 1385-2230, 1386-2187, 1387-2158,1397-2361, 1418-2158, 1435-1848, 1441-1687, 1441-1692, 1441-1953,1444-1800, 1492-1822, 1510-2325, 1510- 2466, 1511-2455, 1514-1849,1547-2385, 1549-2384, 1551-1814, 1552-2312, 1552-2473, 1553-2387,1579-1800, 1581-2438, 1582-1800, 1593-2158, 1593-2481, 1598-2110,1613-2433, 1622-1849, 1638-1800, 1652-2105, 1674- 2321, 1733-2541,1736-2399, 1746-2581, 1760-2274, 1778-2733, 1790-2733, 1848-2230,1848-2400, 1849-2733, 1865-2679, 1880-2735, 1882-2477, 1884-2735,1886-2735, 1889-2733, 1905-2734, 1911-2300, 1925-2185, 1927- 2733,1943-2733, 1944-2733, 1945-2735, 1946-2733, 1946-2735, 1947-2735,1950-2733, 1951-2735, 1953-2733, 1965-2733, 1975-2733, 1978-2274,1978-2737, 1981-2735, 1984-2733, 1987-2733, 1989-2735, 1990-2733, 1991-2733, 1992-2733, 1992-2788, 1993-2880, 2001-2733, 2002-2733, 2002-2897,2004-2733, 2005-2734, 2007-2733, 2008-2733, 2012-2680, 2014-2733,2021-2733, 2025-2906, 2028-2702, 2028-2733, 2052-2296, 2052-2733,2060-2733, 2069-2733, 2072-2680, 2072-2767, 2094-2733, 2099-2733,2105-2733, 2126-2401, 2133-2733, 2139- 2733, 2141-2733, 2161-2986,2165-2946, 2170-2947, 2171-2733, 2172-2986, 2174-2733, 2180-2733,2186-2986, 2194-2729, 2212-2733, 2214-2981, 2214-2986, 2215-2733,2223-2986, 2231-3074, 2234-2733, 2236-2828, 2248-3075, 2253-3075, 2261-3071, 2262-2986, 2264-2986, 2274-2455, 2278-3075, 2282-3075, 2286-2986,2293-3074, 2300-3075, 2327-3075, 2332-3071, 2335-3075, 2338-3075,2345-2736, 2354-3075, 2355-2603, 2369-3074, 2395-2866, 2401-2866,2444-2986, 2453- 3075, 2486-3075, 2536-3075, 2589-3075, 2622-3075,2648-3075 159/7510966CB1 1-1906, 176-570, 459-1414, 480-1280, 480-1281,518-1413, 582-1413, 623-1414, 626-1413, 627-1413, 636-1410, 1906657-1414, 666-1414, 674-937, 766-1414, 815-1325, 817-1414, 820-1204,825-1414, 872-1205, 875-1414, 895-1325, 901-1204, 925-1559, 928-1414,960-1202, 961-1414, 970-1205, 987-1414, 998-1072, 1011-1205, 1049-1452,1068-1205, 1088- 1414, 1112-1583, 1182-1855, 1184-1633, 1481-1901,1485-1633, 1498-1901, 1532-1902 160/7386101CB1/ 1-1887, 6-650, 98-374,99-317, 99-366, 105-375, 369-486, 369-770, 374-750, 377-654, 377-827,377-962, 377-982, 2122 377-984, 377-985, 389-662, 389-807, 389-810,398-818, 406-943, 451-1174, 451-1240, 451-1251, 618-1093, 678- 819,842-1063, 917-1465, 1124-1391, 1339-1884, 1383-1998, 1411-1582,1453-2038, 1485-1938, 1492-1679, 1492-2020, 1498- 2052, 1520-2122,1539-1740, 1604-2056, 1615-2088, 1620-1934, 1625-1896, 1632-2044,1708-1954, 1721-1795, 1747-2008

TABLE 5 Polynucleotide SEQ ID NO: Incyte Project ID: RepresentativeLibrary 81 1417062CB1 MONOTXN03 82 2007701CB1 TESTNOT03 83 2915695CB1THYMFET03 84 2969449CB1 HEAONOT02 85 2994102CB1 KIDNFET02 86 3410251CB1PROSTUS08 87 5330327CB1 SINTFER02 88 5532048CB1 BRAUTDR03 90 60129797CB1BRSTTUT01 91 6246243CB1 TESTNOF01 92 6804755CB1 THYRDIE01 93 6856852CB1BRAIFEN08 95 7493507CB1 THYRNOT03 96 3075994CB1 BONEUNT01 97 2378119CB1BRAFNON02 98 2987418CB1 FIBPFEN06 99 4223862CB1 PANCNOT07 100 6046406CB1BRABDIR02 101 6743529CB1 THP1NOT03 102 7283809CB1 BRAIFEJ01 1037637563CB1 SEMVTDE01 104 7663814CB1 UTRSTME01 105 8001939CB1 LNODTUC02106 8191019CB1 UTRSTMR02 107 919788CB1 KIDNNOT26 108 4758058CB1NERDTDN03 109 7499835CB1 PANCNOT01 110 2484647CB1 THP1AZT01 1112587034CB1 BRAITUT22 112 2702991CB1 BRSTTMT01 113 2744736CB1 PROSUNE04114 2915475CB1 THYMFET03 115 3040427CB1 LSUBNOT03 117 6776909CB1UTRSTMC01 118 7280438CB1 BMARTXE01 119 7499809CB1 IONCDPV07 1207499921CB1 PROSNOT28 121 2705858CB1 PONSAZT01 122 3069892CB1 UTRSNOR01123 3069586CB1 BRAHNOT02 124 7500104CB1 LPARNOT02 125 7500203CB1PANCNOT01 126 4843802CB1 OSTENOT01 127 5877522CB1 BRAHNON05 128617491CB1 PGANNOT01 129 6289901CB1 BRAUTDR03 130 6817709CB1 OVARDIJ01131 6849312CB1 KIDNTMN03 132 7409581CB1 SKINBIT01 133 7437113CB1ADRETUE02 134 7500260CB1 PROSBPS05 135 7659504CB1 PROSNON01 136821165CB1 KERANOT02 137 7499672CB1 THYMNOT04 138 7500276CB1 LEUKNOT02139 1440723CB1 SINTNOR01 140 7479612CB1 OVARTUT07 141 1391514CB1BRAIFER06 142 2102578CB1 UTREDIT07 143 3213122CB1 BRABDIK02 1444326307CB1 BRABNOE02 145 6037749CB1 PITUNOT06 146 6285519CB1 BRAHTDK01147 70336045CB1 EOSIHET02 148 7153577CB1 BONEUNR01 149 7500299CB1KERANOT02 150 7480218CB1 PANCTUT01 152 7501932CB1 OVARDIT06 1547501113CB1 BRSTTUS08 155 7501118CB1 BRSTTUS08 156 7501128CB1 PENITUT01157 7501920CB1 PENITUT01 158 7510325CB1 PENITUT01 159 7510966CB1PROSTUT09 160 7386101CB1 PROSUNE04

TABLE 6 Library Vector Library Description ADRETUE02 PCDNA2.1 This 5′biased random primed library was constructed using RNA isolated fromright adrenal tumor tissue removed from a 49-year-old Caucasian maleduring unilateral adrenalectomy. Pathology indicated adrenal corticalcarcinoma comprising nearly the entire specimen. The tumor was attachedto the adrenal gland which showed mild cortical atrophy. The tumor wasencapsulated, being surrounded by a thin (1-3 mm) rim of connectivetissue. The patient presented with adrenal cancer, abdominal pain,pyrexia of unknown origin, and deficiency anemia. Patient historyincluded benign hypertension. Previous surgeries includedadenotonsillectomy. Patient medications included aspirin, calcium, andiron. Family history included atherosclerotic coronary artery disease inthe mother; cerebrovascular accident and atherosclerotic coronary arterydisease in the father; and benign hypertension in the grandparent(s).BMARTXE01 pINCY This 5′ biased random primed library was constructedusing RNA isolated from treated SH-SY5Y cells derived from a metastaticbone marrow neuroblastoma, removed from a 4-year- old Caucasian female(Schering AG). The medium was MEM/HAM'S F12 with 10% fetal calf serum.After reaching about 80% confluency cells were treated with 6-Hydroxydopamine (6-OHDA) at 100 microM for 8 hours. BONEUNR01 PCDNA2.1This random primed library was constructed using pooled cDNA from twodifferent donors. cDNA was generated using mRNA isolated from anuntreated MG-63 cell line derived from an osteosarcoma tumor removedfrom a 14-year-old Caucasian male (donor A) and using mRNA isolated fromsacral bone tumor tissue removed from an 18-year-old Caucasian female(donor B) during an exploratory laparotomy and soft tissue excision.Pathology indicated giant cell tumor of the sacrum in donor B. Donor B'shistory included pelvic joint pain, constipation, urinary incontinence,unspecified abdominal/pelvic symptoms, and a pelvic soft tissuemalignant neoplasm. Family history included prostate cancer in donor B.BONEUNT01 pINCY Library was constructed using RNA isolated from Saos-2,a primary osteogenic sarcoma cell line (ATCC HTB-85) derived from an11-year-old Caucasian female. BRABDIK02 PSPORT1 This amplified andnormalized library was constructed using pooled cDNA from threedifferent donors. cDNA was generated using mRNA isolated from diseasedvermis tissue removed from a 79-year-old Caucasian female (donor A) whodied from pneumonia, an 83-year-old Caucasian male (donor B) who diedfrom congestive heart failure, and an 87-year-old Caucasian female(donor C) who died from esophageal cancer. Pathology indicated severeAlzheimer's disease in donors A & B and moderate Alzheimer's disease indonor C. Patient history included glaucoma, pseudophakia, gastritis withgastrointestinal bleeding, peripheral vascular disease, chronicobstructive pulmonary disease, seizures, tobacco abuse in remission, andtransitory ischemic attacks in donor A; Parkinson's disease andatherosclerosis in donor B; hypertension, coronary artery disease,cerebral vascular accident, and hypothyroidism in donor C. Familyhistory included Alzheimer's disease in the mother and sibling(s) ofdonor A. Independent clones from this amplified library were normalizedin one round using conditions adapted Soares et al., PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research 6 (1996): 791, except thata significantly longer (48 hours/round) reannealing hybridization wasused. BRABDIR02 pINCY This random primed library was constructed usingRNA isolated from diseased cerebellum tissue removed from the brain of a57-year-old Caucasian male who died from a cerebrovascular accident.Serologies were negative. Patient history included Huntington's disease,emphysema, and tobacco abuse (3-4 packs per day for 40 years). BRABNOE02PBK-CMV This 5′ biased random primed library was constructed using RNAisolated from vermis tissue removed from a 35-year-old Caucasian malewho died from cardiac failure. Pathology indicated moderateleptomeningeal fibrosis and multiple microinfarctions of the cerebralneocortex. Patient history included dilated cardiomyopathy, congestiveheart failure, cardiomegaly, and an enlarged spleen and liver. Patientmedications included simethicone, Lasix, Digoxin, Colace, Zantac,captopril, and Vasotec. BRAFNON02 pINCY This normalized frontal cortextissue library was constructed from 10.6 million independent clones froma frontal cortex tissue library. Starting RNA was made from superiorfrontal cortex tissue removed from a 35-year-old Caucasian male who diedfrom cardiac failure. Pathology indicated moderate leptomeningealfibrosis and multiple microinfarctions of the cerebral neocortex.Grossly, the brain regions examined and cranial nerves wereunremarkable. No atherosclerosis of the major vessels was noted.Microscopically, the cerebral hemisphere revealed moderate fibrosis ofthe leptomeninges with focal calcifications. There was evidence ofshrunken and slightly eosinophilic pyramidal neurons throughout thecerebral hemispheres. There were also multiple small microscopic areasof cavitation with surrounding gliosis scattered throughout the cerebralcortex. Patient history included dilated cardiomyopathy, congestiveheart failure, cardiomegaly, and an enlarged spleen and liver. Patientmedications included simethicone, Lasix, Digoxin, Colace, Zantac,captopril, and Vasotec. The library was normalized in two rounds usingconditions adapted from Soares et al., PNAS (1994) 91: 9228 and Bonaldoet al., Genome Research (1996) 6: 791, except that a significantlylonger (48 hours/round) reannealing hybridization was used. BRAHNON05pINCY This normalized hippocampus tissue library was constructed from1.6 million independent clones from a hippocampus tissue library.Starting RNA was made from posterior hippocampus removed from a35-year-old Caucasian male who died from cardiac failure. Pathologyindicated moderate leptomeningeal fibrosis and multiple microinfarctionsof the cerebral neocortex. The cerebral hemisphere revealed moderatefibrosis of the leptomeninges with focal calcifications. There wasevidence of shrunken and slightly eosinophilic pyramidal neuronsthroughout the cerebral hemispheres. There were small microscopic areasof cavitation with gliosis, scattered through the cerebral cortex.Patient history included cardiomyopathy, CHF, cardiomegaly, an enlargedspleen and liver. Patient medications included simethicone, Lasix,Digoxin, Colace, Zantac, captopril, and Vasotec. The library wasnormalized in two rounds using conditions adapted from Soares et al.,PNAS (1994) 91: 9228 and Bonaldo et al., Genome Research 6 (1996): 791,except that a significantly longer (48 hours/round) reannealinghybridization was used. BRAHNOT02 pINCY Library was constructed usingRNA isolated from posterior hippocampus tissue removed from an81-year-old Caucasian female who died from a hemorrhage and rupturedthoracic aorta due to atherosclerosis. Pathology indicated moderateatherosclerosis involving the internal carotids, bilaterally;microscopic infarcts of the frontal cortex and hippocampus; andscattered diffuse amyloid plaques and neurofibrillary tangles,consistent with age. The posterior hippocampus contained a microscopicarea of cystic cavitation with hemosiderin-laden macrophages surroundedby reactive gliosis. The patient presented with sepsis, cholangitis, andpost-operative atelectasis and pneumonia. Patient history included CAD,cardiomegaly due to left ventricular hypertrophy, splenomegaly,arteriolonephrosclerosis, nodular colloidal goiter, emphysema,congestive heart failure, hypothyroidism, and peripheral vasculardisease. Previous surgeries included cholecystectomy and Bilroth Igastrectomy for ulcer. Patient medications included Lasix, Synthroid,Pancrease, Voltaren, Vicoden, Zantac and K-Dur. BRAHTDK01 PSPORT1 Thisamplified and normalized library was constructed using pooled RNAisolated from archaecortex, anterior and posterior hippocampus tissueremoved from a 55-year-old Caucasian female who died fromcholangiocarcinoma. Pathology indicated mild meningeal fibrosispredominately over the convexities, scattered axonal spheroids in thewhite matter of the cingulate cortex and the thalamus, and a fewscattered neurofibrillary tangles in the entorhinal cortex and theperiaqueductal gray region. Pathology for the associated tumor tissueindicated well-differentiated cholangiocarcinoma of the liver withresidual or relapsed tumor. Patient history included cholangiocarcinoma,post-operative Budd-Chiari syndrome, biliary ascites, hydrothorax,dehydration, malnutrition, oliguria and acute renal failure. Previoussurgeries included cholecystectomy and resection of 85% of the liver.7.6 × 10e5 independent clones from this amplified library werenormalized in 1 round using conditions adapted Soares et al., PNAS(1994) 91: 9228-9232 and Bonaldo et al., Genome Research (1996) 6: 791,except that a significantly longer (48 hours/round) reannealinghybridization was used. BRAIFEJ01 pRARE This random primed 5′ capisolated library was constructed using RNA isolated from brain tissueremoved from a Caucasian male fetus who died at 23 weeks' gestation frompremature birth. Serologies were negative. Family history includeddiabetes in the mother. BRAIFEN08 pINCY This normalized fetal braintissue library was constructed from 400 thousand independent clones froma fetal brain tissue library. Starting RNA was made from brain tissueremoved from a Caucasian male fetus who was stillborn with a hypoplasticleft heart at 23 weeks' gestation. The library was normalized in 2rounds using conditions adapted from Soares et al., PNAS (1994) 91: 9228and Bonaldo et al., Genome Research (1996) 6: 791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. BRAIFER06 PCDNA2.1 This random primed library was constructedusing RNA isolated from brain tissue removed from a Caucasian male fetuswho was stillborn with a hypoplastic left heart at 23 weeks' gestation.Serologies were negative. BRAITUT22 pINCY Library was constructed usingRNA isolated from brain tumor tissue removed from the rightfrontal/parietal lobe of a 76- year-old Caucasian female during excisionof a cerebral meningeal lesion. Pathology indicated a meningioma. Familyhistory included senile dementia. BRAUTDR03 PCDNA2.1 This random primedlibrary was constructed using RNA isolated from pooled globus pallidusand substantia innominata tissue removed from a 55-year-old Caucasianfemale who died from cholangiocarcinoma. Pathology indicated mildmeningeal fibrosis predominately over the convexities, scattered axonalspheroids in the white matter of the cingulate cortex and the thalamus,and a few scattered neurofibrillary tangles in the entorhinal cortex andthe periaqueductal gray region. Pathology for the associated tumortissue indicated well-differentiated cholangiocarcinoma of the liverwith residual or relapsed tumor. Patient history includedcholangiocarcinoma, post-operative Budd-Chiari syndrome, biliaryascites, hydrothorax, dehydration, malnutrition, oliguria and acuterenal failure. Previous surgeries included cholecystectomy and resectionof 85% of the liver. BRSTTMT01 pINCY Library was constructed using RNAisolated from breast tissue removed from a 43-year-old Caucasian femaleduring a unilateral extended simple mastectomy. Pathology for theassociated tumor tissue indicated recurrent grade 4, nuclear grade 3,ductal carcinoma. Angiolymphatic space invasion was identified. Leftbreast needle biopsy indicated grade 4 ductal adenocarcinoma. Paraffinembedded tissue was estrogen positive. Patient history included breastcancer and deficiency anemia. Family history included cervical cancer.BRSTTUS08 pINCY This subtracted library was constructed using 2.36 Mclones from a breast tumor library and was subjected to two rounds ofsubtraction hybridization with 2.32 M clones from a prostate tissuelibrary. RNA was isolated from breast tumor tissue removed from theright breast of a 46-year-old Caucasian female during a unilateralextended simple mastectomy with breast reconstruction. Pathologyindicated an invasive grade 3 adenocarcinoma. Patient history includedbreast cancer. Subtractive hybridization conditions were based on themethodologies of Swaroop et al. NAR (1991) 19: 1954 and Bonaldo et al.Genome Research (1996) 6: 791. BRSTTUT01 PSPORT1 Library was constructedusing RNA isolated from breast tumor tissue removed from a 55-year-oldCaucasian female during a unilateral extended simple mastectomy.Pathology indicated invasive grade 4 mammary adenocarcinoma of mixedlobular and ductal type, extensively involving the left breast. Thetumor was identified in the deep dermis near the lactiferous ducts withextracapsular extension. Seven mid and low and five high axillary lymphnodes were positive for tumor. Proliferative fibrocysytic changes werecharacterized by apocrine metaplasia, sclerosing adenosis, cystformation, and ductal hyperplasia without atypia. Patient historyincluded atrial tachycardia, blood in the stool, and a benign breastneoplasm. Family history included benign hypertension, atheroscleroticcoronary artery disease, cerebrovascular disease, and depressivedisorder. EOSIHET02 PBLUESCRIPT Library was constructed using RNAisolated from peripheral blood cells apheresed from a 48-year-oldCaucasian male. Patient history included hypereosinophilia. The cellpopulation was determined to be greater than 77% eosinophils by Wright'sstaining. FIBPFEN06 pINCY The normalized prostate stromal fibroblasttissue libraries were constructed from 1.56 million independent clonesfrom a prostate fibroblast library. Starting RNA was made fromfibroblasts of prostate stroma removed from a male fetus, who died after26 weeks' gestation. The libraries were normalized in two rounds usingconditions adapted from Soares et al., PNAS (1994) 91: 9228 and Bonaldoet al., Genome Research (1996) 6: 791, except that a significantlylonger (48- hours/round)reannealing hybridization was used. The librarywas then linearized and recircularized to select for insert containingclones as follows: plasmid DNA was prepped from approximately 1 millionclones from the normalized prostate stromal fibroblast tissue librariesfollowing soft agar transformation. HEAONOT02 pINCY Library wasconstructed using RNA isolated from aortic tissue removed from a10-year-old Caucasian male, who died from anoxia. IONCDPV07 PCR2-TOPOTALibrary was constructed using pooled cDNA from different donors. cDNAwas generated using mRNA isolated from pooled skeletal muscle tissueremoved from ten 21 to 57- year-old Caucasian male and female donors whodied from sudden death; from pooled thymus tissue removed from nine 18to 32-year-old Caucasian male and female donors who died from suddendeath; from pooled liver tissue removed from 32 Caucasian male andfemale fetuses who died at 18-24 weeks gestation due to spontaneousabortion; from kidney tissue removed from 59 Caucasian male and femalefetuses who died at 20-33 weeks gestation due to spontaneous abortion;and from brain tissue removed from a Caucasian male fetus who died at 23weeks gestation due to fetal demise. KERANOT02 PSPORT1 Library wasconstructed using RNA isolated from epidermal breast keratinocytes(NHEK). NHEK (Clontech #CC-2501) is a human breast keratinocyte cellline derived from a 30- year-old black female during breast-reductionsurgery. KIDNFET02 pINCY Library was constructed using RNA isolated fromkidney tissue removed from a Caucasian male fetus, who was stillbornwith a hypoplastic left heart and died at 23 weeks' gestation. KIDNNOT26pINCY Library was constructed using RNA isolated from left kidneymedulla and cortex tissue removed from a 53-year-old Caucasian femaleduring a nephroureterectomy. Pathology for the associated tumor tissueindicated grade 2 renal cell carcinoma involving the lower pole of thekidney. Patient history included hyperlipidemia, cardiac dysrhythmia,metrorrhagia, normal delivery, cerebrovascular disease, atheroscleroticcoronary artery disease, and tobacco abuse. Family history includedcerebrovascular disease and atherosclerotic coronary artery disease.KIDNTMN03 pINCY This normalized kidney tissue library was constructedfrom 2.08 million independent clones from a pool of two libraries fromtwo different donors. Starting RNA was made from right kidney tissueremoved from an 8-year-old Caucasian female (donor A) who died from amotor vehicle accident and left kidney medulla and cortex tissue removedfrom a 53-year-old Caucasian female (donor B) during anephroureterectomy. In donor B, pathology for the matched tumor tissueindicated grade 2 renal cell carcinoma involving the lower pole of thekidney. Medical history included hyperlipidemia, cardiac dysrhythmia,metrorrhagia, normal delivery, cerebrovascular disease, andatherosclerotic coronary artery disease in donor B. The library wasnormalized in two rounds using conditions adapted from Soares et al.,PNAS (1994) 91: 9228-9232 and Bonaldo et al., Genome Research 6 (1996):791, except that a significantly longer (48 hours/round) reannealinghybridization was used. LEUKNOT02 pINCY Library was constructed usingRNA isolated from white blood cells of a 45-year-old female with bloodtype O+. The donor tested positive for cytomegalovirus (CMV). LNODTUC02pINCY This large size fractionated library was constructed using pooledcDNA from two donors. cDNA was generated using mRNA isolated from pelviclymph node tumor tissue removed from a 42-year-old Caucasian female(donor A) during regional lymph node excision and removal of a solitaryovary and from left axillary lymph node tumor tissue from another donor(donor B). For donor A, pathology indicated Hodgkin's disease, nodularsclerosing type. The cells were reactive for CD15 (Leu-MI). The patientpresented with nodular lymphoma and unspecified abdominal and pelvicsymptoms. Patient history included diabetes during pregnancy and normaldelivery. Previous surgeries included bilateral breast implants,appendectomy, bilateral tubal destruction and dilation and curettage.Patient medications included methylprednisone, Cefclor, and Naproxen.Family history included atherosclerotic coronary artery disease in thefather and alcohol abuse in remission in the sibling. For donor B,pathology indicated metastatic adenocarcinoma. LPARNOT02 pINCY Librarywas constructed using RNA isolated from tissue obtained from the leftparotid (salivary) gland of a 70-year-old male with parotid cancer.LSUBNOT03 pINCY Library was constructed using RNA isolated fromsubmandibular gland tissue obtained from a 68-year-old Caucasian maleduring a sialoadenectomy. Family history included acute myocardialinfarction, atherosclerotic coronary artery disease, and type IIdiabetes. MONOTXN03 pINCY Normalized, treated monocyte tissue librarywas constructed from 7.6 million independent clones from a treatedmonocyte library. Starting RNA was made from RNA isolated from treatedmonocytes from peripheral blood obtained from a 42-year old female. Thecells were treated with anti-interleukin-10 (anti-IL-10) andlipopolysaccharide (LPS). The anti-IL-10 was added at time 0 at 10 ng/mland LPS was added at 1 hour at 5 ng/ml. The monocytes were isolated frombuffy coat by adherence to plastic. Incubation time was 24 hours. CDNAsynthesis was initiated using a NotI-anchored oligo(dT) primer. Thelibraries were normalized in two rounds using conditions adapted fromSoares et al., PNAS (1994) 91: 9228 and Bonaldo et al., Genome Research(1996 6): 791, except that a significantly longer (48 -hours/round)reannealing hybridization was used. The libraries were then linearizedand recircularized to select for insert containing clones as follows:plasmid DNA was prepped from approximately 1 million clones from thenormalized, treated monocyte tissue libraries following soft agartransformation. The DNA was linearized with NotI and insert containingclones were size-selected by agarose gel electrophoresis andrecircularized by ligation. NERDTDN03 pINCY This normalized dorsal rootganglion tissue library was constructed from 1.05 million independentclones from a dorsal root ganglion tissue library. Starting RNA was madefrom dorsal root ganglion tissue removed from the cervical spine of a32-year-old Caucasian male who died from acute pulmonary edema, acutebronchopneumonia, bilateral pleural effusions, pericardial effusion, andmalignant lymphoma (natural killer cell type). The patient presentedwith pyrexia of unknown origin, malaise, fatigue, and gastrointestinalbleeding. Patient history included probable cytomegalovirus infection,liver congestion, and steatosis, splenomegaly, hemorrhagic cystitis,thyroid hemorrhage, respiratory failure, pneumonia of the left lung,natural killer cell lymphoma of the pharynx, Bell's palsy, and tobaccoand alcohol abuse. Previous surgeries included colonoscopy, closed colonbiopsy, adenotonsillectomy, and nasopharyngeal endoscopy and biopsy.Patient medications included Diflucan (fluconazole), Deltasone(prednisone), hydrocodone, Lortab, Alprazolam, Reazodone,ProMace-Cytabom, Etoposide, Cisplatin, Cytarabine, and dexamethasone.The patient received radiation therapy and multiple blood transfusions.The library was normalized in 2 rounds using conditions adapted fromSoares et al., PNAS (1994) 91: 9228-9232 and Bonaldo et al., GenomeResearch 6 (1996): 791, except that a significantly longer (48hours/round) reannealing hybridization was used. OSTENOT01 pINCY Librarywas constructed using RNA isolated from untreated osteoblasts removedfrom the clavicle of a 40-year-old male. OVARDIJ01 pIGEN This randomprimed 5′ cap isolated library was constructed using RNA isolated fromdiseased right ovary tissue removed from a 47-year-old Caucasian femaleduring total abdominal hysterectomy, dilation and curettage, bilateralsalpingo- oophorectomy, repair of ureter, and incidental appendectomy.Pathology indicated endometriosis. Pathology for the associated tumortissue indicated multiple leiomyomata. The left ovary contained a corpusluteum. There was endometriosis involving the posterior serosa. Thepatient presented with metrorrhagia and a benign neoplasm of the ovary.Patient history included normal delivery, joint pain in multiple joints,and unilateral congenital hip dislocation. Previous surgeries includedtotal hip replacement. Patient medications included calcium. Familyhistory included kidney cancer in the mother; atherosclerotic coronaryartery disease and aortocoronary bypass of 3 coronary arteries in thefather; benign hypertension and Hodgkin's disease in the sibling(s); andbenign hypertension and cerebrovascular accident in the grandparent(s).OVARDIT06 pINCY The library was constructed using RNA isolated fromdiseased left ovarian tissue removed from a 24-year-old Caucasian femaleduring left ovary lesion excision. Pathology indicated endometriosis(endometrioma) of the left ovary, consisting of a tan-maroon collapsedcyst. The serosal surface was tan-purple and irregular with fibrous andfibrinous adhesions. The internal surface was maroon-green and ulceratedwith no papillary excrescences. Microscopic sections revealed fragmentsof ovarian stroma associated with focal areas with numerous macrophagescontaining hemosiderin pigment, fibrosis and rare glands surrounded byendometrial stroma. The patient presented with pain, dysmenorrhea, and apelvic mass. OVARTUT07 pINCY Library was constructed using RNA isolatedfrom right ovarian tumor tissue removed from a 58-year-old Caucasianfemale during bilateral salpingo-oophorectomy, regional lymph nodeexcision, destruction of peritoneal tissue, cystocele repair, and skinrepair. Pathology indicated FIGO (International Federation of Gynecologyand Obstetrics) grade 3 adenocarcinoma, serous type, forming a mass andentirely replacing the right ovary. The left pelvic sidewall revealed amicroscopic focus of metastatic adenocarcinoma. Patient history includedhyperlipidemia, thrombophlebitis, and carcinoma in situ of the cervixuteri. Family history included cerebrovascular disease, breast cancer,hyperlipidemia, atherosclerotic coronary artery disease, and heartfailure. PANCNOT01 PBLUESCRIPT Library was constructed using RNAisolated from the pancreatic tissue of a 29-year-old Caucasian male whodied from head trauma. PANCNOT07 pINCY Library was constructed using RNAisolated from the pancreatic tissue of a Caucasian male fetus, who diedat 23 weeks' gestation. PANCTUT01 pINCY Library was constructed usingRNA isolated from pancreatic tumor tissue removed from a 65-year-oldCaucasian female during radical subtotal pancreatectomy. Pathologyindicated an invasive grade 2 adenocarcinoma. Patient history includedtype II diabetes, osteoarthritis, cardiovascular disease, benignneoplasm in the large bowel, and a cataract. Previous surgeries includeda total splenectomy, cholecystectomy, and abdominal hysterectomy. Familyhistory included cardiovascular disease, type II diabetes, and stomachcancer. PENITUT01 pINCY Library was constructed using RNA isolated fromtumor tissue removed from the penis of a 64-year-old Caucasian maleduring penile amputation. Pathology indicated a fungating invasive grade4 squamous cell carcinoma involving the inner wall of the foreskin andextending onto the glans penis. Patient history included benign neoplasmof the large bowel, atherosclerotic coronary artery disease, anginapectoris, gout, and obesity. Family history included malignantpharyngeal neoplasm, chronic lymphocytic leukemia, and chronic liverdisease. PGANNOT01 PSPORT1 Library was constructed using RNA isolatedfrom paraganglionic tumor tissue removed from the intra-abdominal regionof a 46-year-old Caucasian male during exploratory laparotomy. Pathologyindicated a benign paraganglioma and was associated with a grade 2 renalcell carcinoma, clear cell type, which did not penetrate the capsule.Surgical margins were negative for tumor. PITUNOT06 pINCY Library wasconstructed using RNA isolated from pituitary gland tissue removed froma 55-year-old male who died from chronic obstructive pulmonary disease.Neuropathology indicated there were no gross abnormalities, other thanmild ventricular enlargement. There was no apparent microscopicabnormality in any of the neocortical areas examined, except for anumber of silver positive neurons with apical dendrite staining,particularly in the frontal lobe. The significance of this wasundetermined. The only other microscopic abnormality was that there wasprominent silver staining with some swollen axons in the CA3 region ofthe anterior and posterior hippocampus. Microscopic sections of thecerebellum revealed mild Bergmann's gliosis in the Purkinje cell layer.Patient history included schizophrenia. PONSAZT01 pINCY Library wasconstructed using RNA isolated from diseased pons tissue removed fromthe brain of a 74-year-old Caucasian male who died from Alzheimer'sdisease. PROSBPS05 pINCY This subtracted prostate tissue library wasconstructed using 4.48 × 10e5 clones from diseased prostate tissue andwas subjected to two rounds of subtraction hybridization with 1.56million clones from a breast tissue library. The starting library forsubtraction was constructed using RNA isolated from diseased prostatetissue removed from a 70-year-old Caucasian male during a radicalprostatectomy and closed prostatic biopsy. Pathology indicated benignprostatic hypertrophy. Pathology for the matched tumor tissue indicatedadenocarcinoma. The patient presented with elevated prostate specificantigen and induration. Patient history included benign hypertension,gastrointestinal bleed, cardiac dysrhythmia, cardiac arrest,hyperlipidemia, alcohol abuse and fractured mandible. Previous surgeriesincluded splenectomy, cholecystectomy and inguinal hernia repair.Patient medications included Verapamil and antacids. Family historyincluded benign hypertension, myocardial infarction and coronaryatherosclerosis in the mother; tobacco abuse and lung cancer in thefather; tobacco abuse, cerebrovascular accident and lung cancer in thesibling(s). The hybridization probe for subtraction was derived from asimilarly constructed library from RNA isolated from nontumorous breasttissue from a different donor. Subtractive hybridization conditions werebased on the methodologies of Swaroop et al., NAR 19 (1991): 1954 andBonaldo, et al. Genome Research 6 (1996): 791. PROSNON01 PSPORT1 Thisnormalized prostate library was constructed from 4.4 M independentclones from a prostate library. Starting RNA was made from prostatetissue removed from a 28-year-old Caucasian male who died from aself-inflicted gunshot wound. The normalization and hybridizationconditions were adapted from Soares, M. B. et al. (1994) Proc. Natl.Acad. Sci. USA 91: 9228-9232, using a longer (19 hour) reannealinghybridization period. PROSNOT28 pINCY Library was constructed using RNAisolated from diseased prostate tissue removed from a 55-year-oldCaucasian male during a radical prostatectomy and regional lymph nodeexcision. Pathology indicated adenofibromatous hyperplasia. Pathologyfor the associated tumor tissue indicated adenocarcinoma, Gleason grade5 + 4. The patient presented with elevated prostate specific antigen(PSA). Family history included lung and breast cancer. PROSTUS08 pINCY.This subtracted prostate tumor library was constructed using 2.36million clones from a prostate tumor library and was subjected to oneround of subtractive hybridization with 448,000 clones from a prostatetumor library. The starting library for subtraction was constructedusing RNA isolated from a prostate tumor removed from a 59-year-oldCaucasian male during a radical prostatectomy with regional lymph nodeexcision. Pathology indicated adenocarcinoma (Gleason grade 3 + 3)Adenofibromatous hyperplasia was present. The patient presented withelevated prostate-specific antigen (PSA). Patient history included colondiverticuli, asbestosis, and thrombophlebitis. Family history includedmultiple myeloma, hyperlipidemia, and rheumatoid arthritis. Subtractivehybridization conditions were based on the methodologies of Swaroop etal., NAR (1991) 19: 1954 and Bonaldo, et al. Genome Research (1996) 6:791. PROSTUT09 pINCY Library was constructed using RNA isolated fromprostate tumor tissue removed from a 66-year-old Caucasian male during aradical prostatectomy, radical cystectomy, and urinary diversion.Pathology indicated grade 3 transitional cell carcinoma. The patientpresented with prostatic inflammatory disease. Patient history includedlung neoplasm, and benign hypertension. Family history included amalignant breast neoplasm, tuberculosis, cerebrovascular disease,atherosclerotic coronary artery disease and lung cancer. PROSUNE04 pINCYThis 5′ biased random primed library was constructed using RNA isolatedfrom an untreated LNCaP cell line, derived from prostate carcinoma withmetastasis to the left supraclavicular lymph nodes, removed from a50-year-old Caucasian male (Schering). SEMVTDE01 PCDNA2.1 This 5′ biasedrandom primed library was constructed using RNA isolated from seminalvesicle tissue removed from a 63- year-old Caucasian male during closedprostatic biopsy, radical prostatectomy, and regional lymph nodeexcision. Pathology for the associated tumor tissue indicated Gleasongrade 2 + 3 adenocarcinoma in the right side of the prostate.Adenofibromatous hyperplasia was present. The patient presented withprostate cancer, elevated prostate specific antigen and prostatichyperplasia. Patient history included kidney calculus, extrinsic asthma,benign bowel neoplasm, backache, tremor, and tobacco abuse in remission.Previous surgeries included adenotonsillectomy. Patient medicationsincluded Ventolin and Vanceril. Family history included atheroscleroticcoronary artery disease and acute myocardial infarction in the mother;atherosclerotic coronary artery disease and acute myocardial infarctionin the father; and stomach cancer and extrinsic asthma in thegrandparent(s). SINTFER02 pINCY This random primed library wasconstructed using RNA isolated from small intestine tissue removed froma Caucasian male fetus who died from fetal demise. SINTNOR01 PCDNA2.1This random primed library was constructed using RNA isolated from smallintestine tissue removed from a 31-year-old Caucasian female duringRoux-en-Y gastric bypass. Patient history included clinical obesity.SKINBIT01 pINCY Library was constructed using RNA isolated from diseasedskin tissue of the left lower leg. Patient history included erythemanodosum of the left lower leg. TESTNOF01 PSPORT1 This 5′ cap isolatedfull-length library was constructed using RNA isolated from testistissue removed from a 26-year-old Caucasian male who died from headtrauma due to a motor vehicle accident. Serologies were negative.Patient history included a hernia at birth, tobacco use (1½ ppd),marijuana use, and daily alcohol use (beer and hard liquor). TESTNOT03PBLUESCRIPT Library was constructed using RNA isolated from testiculartissue removed from a 37-year-old Caucasian male, who died from liverdisease. Patient history included cirrhosis, jaundice, and liverfailure. THP1AZT01 pINCY Library was constructed using RNA isolated fromTHP-1 promonocyte cells treated for three days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is a human promonocyte linederived from peripheral blood of a 1-year-old Caucasian male with acutemonocytic leukemia ( Int. J. Cancer (1980) 26: 171). THP1NOT03 pINCYLibrary was constructed using RNA isolated from untreated THP-1 cells.THP-1 is a human promonocyte line derived from the peripheral blood of a1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J.Cancer (1980) 26: 171). THYMFET03 pINCY Library was constructed usingRNA isolated from thymus tissue removed from a Caucasian male fetus.THYMNOT04 pINCY Library was constructed using RNA isolated from thymustissue removed from a 3-year-old Caucasian male, who died from anoxia.Serologies were negative. The patient was not taking any medications.THYRDIE01 PCDNA2.1 This 5′ biased random primed library was constructedusing RNA isolated from diseased thyroid tissue removed from a 22-year-old Caucasian female during closed thyroid biopsy, partialthyroidectomy, and regional lymph node excision. Pathology indicatedadenomatous hyperplasia. The patient presented with malignant neoplasmof the thyroid. Patient history included normal delivery, alcohol abuse,and tobacco abuse. Previous surgeries included myringotomy. Patientmedications included an unspecified type of birth control pills. Familyhistory included hyperlipidemia and depressive disorder in the mother;and benign hypertension, congestive heart failure, and chronic leukemiain the grandparent(s). THYRNOT03 pINCY Library was constructed using RNAisolated from thyroid tissue removed from the left thyroid of a28-year-old Caucasian female during a complete thyroidectomy. Pathologyindicated a small nodule of adenomatous hyperplasia present in the leftthyroid. Pathology for the associated tumor tissue indicated dominantfollicular adenoma, forming a well-encapsulated mass in the leftthyroid. UTREDIT07 pINCY Library was constructed using RNA isolated fromdiseased endometrial tissue removed from a female during endometrialbiopsy. Pathology indicated in phase endometrium with missing beta 3,Type II defects. UTRSNOR01 pINCY Library was constructed using RNAisolated from uterine endometrium tissue removed from a 29-year-oldCaucasian female during a vaginal hysterectomy and cystocele repair.Pathology indicated the endometrium was secretory, and the cervix showedmild chronic cervicitis with focal squamous metaplasia. Pathology forthe associated tumor tissue indicated intramural uterine leiomyoma.Patient history included hypothyroidism, pelvic floor relaxation, andparaplegia. Family history included benign hypertension, type IIdiabetes, and hyperlipidemia. UTRSTMC01 PSPORT1 This large sizefractionated library was constructed using pooled cDNA from two donors.cDNA was generated using mRNA isolated from uterus tissue removed from a49-year-old Caucasian female (donor A) during vaginal hysterectomy andbilateral salpingo-oophorectomy and from uterus tissue removed from a55-year-old Caucasian female (donor B) during vaginal hysterectomy andbilateral salpingo-oophorectomy. For donor A, pathology indicatedinactive endometrium and cervix with no diagnostic changes. Pathologyfor the matched tumor tissue indicated multiple (6) intramuralleiomyomata. The patient presented with excessive menstruation,deficiency anemia, and dysmenorrhea. Patient history included abdominalpregnancy, headache, and chronic obstructive asthma. Previous surgeriesincluded hemorrhoidectomy, knee ligament repair, and intranasal lesiondestruction. Patient medications included Azmacort, Proventil,Trazadone, Zostrix HP, iron, Premarin, and vitamin C. Family historyincluded alcohol abuse, atherosclerotic coronary artery disease, upperlobe lung cancer, and carotid endarterectomy in the father; breastfibroadenosis in the sibling(s); and acute myocardial infarction, livercancer, acute leukemia, and breast cancer (central) in thegrandparent(s). For donor B, pathology indicated proliferativeendometrium and unremarkable cervix. The patient presented withexcessive menstruation, pelvic pain, uterine leiomyoma, andendometriosis. Patient history included hypothyroid, normal delivery,bladder dilation, irritable colon, and endometrial hyperplasia. Previoussurgeries included adenotonsillectomy. Patient medications includedSynthroid and vitamins. Family history included atherosclerotic coronaryartery disease and malignant breast neoplasm in the mother; malignantcolon neoplasm and aterial embolism in the father; and drug abuse in thesibling(s). UTRSTME01 PCDNA2.1 This 5′ biased random primed library wasconstructed using RNA isolated from uterus tissue removed from a49-year-old Caucasian female during vaginal hysterectomy and bilateralsalpingo-oophorectomy. Pathology for the matched tumor tissue indicatedmultiple (6) intramural leiomyomata. The patient presented withexcessive menstruation, deficiency anemia, and dysmenorrhea. Patienthistory included abdominal pregnancy, headache, and chronic obstructiveasthma. Previous surgeries included hemorrhoidectomy, knee ligamentrepair, and intranasal lesion destruction. Patient medications includedAzmacort, Proventil, Trazadone, Zostrix HP, iron, Premarin, and vitaminC. Family history included alcohol abuse, atherosclerotic coronaryartery disease, upper lobe lung cancer, and carotid endarterectomy inthe father; breast fibroadenosis in the sibling(s); and acute myocardialinfarction, liver cancer, acute leukemia, and breast cancer (central) inthe grandparent(s). UTRSTMR02 PCDNA2.1 This random primed library wasconstructed using pooled cDNA from two different donors. cDNA wasgenerated using mRNA isolated from endometrial tissue removed from a32-year- old female (donor A) and using mRNA isolated from myometriumremoved from a 45-year-old female (donor B) during vaginal hysterectomyand bilateral salpingo- oophorectomy. In donor A, pathology indicatedthe endometrium was secretory phase. The cervix showed severe dysplasia(CIN III) focally involving the squamocolumnar junction at the 1, 6 and7 o'clock positions. Mild koilocytotic dysplasia was also identifiedwithin the cervix. In donor B, pathology for the matched tumor tissueindicated multiple (23) subserosal, intramural, and submucosalleiomyomata. Patient history included stress incontinence, extrinsicasthma without status asthmaticus and normal delivery in donor B. Familyhistory included cerebrovascular disease, depression, andatherosclerotic coronary artery disease in donor B.

TABLE 7 Program Description Reference Parameter Threshold ABI FACTURA Aprogram that removes vector sequences and masks Applied Biosystems,ambiguous bases in nucleic acid sequences. Foster City, CA. ABI/PARACELA Fast Data Finder useful in comparing and Applied Biosystems, Mismatch< 50% FDF annotating amino acid or nucleic acid sequences. Foster City,CA; Paracel Inc., Pasadena, CA. ABI A program that assembles nucleicacid sequences. Applied Biosystems, AutoAssembler Foster City, CA. BLASTA Basic Local Alignment Search Tool useful in Altschul, S. F. et al.ESTs: Probability sequence similarity search for amino acid and (1990)J. Mol. Biol. value = 1.0E−8 nucleic acid sequences. BLAST includes five215: 403-410; or less; Full functions: blastp, blastn, blastx, tblastn,Altschul, S. F. et al. Length sequences: and tblastx. (1997) NucleicAcids Probability value = Res. 25: 3389-3402. 1.0E−10 or less FASTA APearson and Lipman algorithm that searches Pearson, W. R. and ESTs:fasta E for similarity between a query sequence and D. J. Lipman (1988)value = 1.06E−6; a group of sequences of the same type. Proc. Natl. AcadSci. Assembled ESTs: FASTA comprises as least five functions: USA 85:2444-2448; fasta Identity = fasta, tfasta, fastx, tfastx, and Pearson,W. R. (1990) 95% or greater ssearch. Methods Enzymol. 183: and Match63-98; and Smith, length = 200 T. F. and M. S. Waterman bases orgreater; (1981) Adv. Appl. Math. fastx E value = 2: 482-489. 1.0E−8 orless; Full Length sequences: fastx score = 100 or greater BLIMPS ABLocks IMProved Searcher that matches a Henikoff, S. and J. G.Probability sequence against those in BLOCKS, PRINTS, Henikoff (1991)value = 1.0E−3 or DOMO, PRODOM, and PFAM databases to search NucleicAcids Res. 19: less for gene families, sequence homology, and 6565-6572;Henikoff, structural fingerprint regions. J. G. and S. Henikoff (1996)Methods Enzymol. 266: 88-105; and Attwood, T. K. et al. (1997) J. Chem.Inf. Comput. Sci. 37: 417-424. HMMER An algorithm for searching a querysequence Krogh, A. et al. (1994) PFAM, INCY, SMART or against hiddenMarkov model (HMM)-based J. Mol. Biol. TIGRFAM hits: Probabilitydatabases of protein family consensus sequences, 235: 1501-1531; value =1.0E−3 such as PFAM, INCY, SMART and TIGRFAM. Sonnhammer, E. L. L. orless; Signal et al. (1988) Nucleic peptide hits: Acids Res. 26: 320-322;Score = 0 or greater Durbin, R. et al. (1998) Our World View, in aNutshell, Cambridge Univ. Press, pp. 1-350. ProfileScan An algorithmthat searches for structural and Gribskov, M. et al. Normalized qualitysequence motifs in protein sequences that match (1988) CABIOS 4: 61-66;score ≦ GCG sequence patterns defined in Prosite. Gribskov, M. et al.specified “HIGH” (1989) Methods value for that Enzymol. 183: 146-159;particular Prosite Bairoch, A. et al. motif. (1997) Nucleic Acids Res.Generally, score = 25: 217-221. 1.4-2.1. Phred A base-calling algorithmthat examines automated Ewing, B. et al. (1998) sequencer traces withhigh sensitivity and Genome Res. 8: 175-185; probability. Ewing, B. andP. Green (1998) Genome Res. 8: 186-194. Phrap A Phils Revised AssemblyProgram including Smith, T. F. and M. S. Score = 120 or SWAT andCrossMatch, programs based on efficient Waterman (1981) Adv. greater;Match implementation of the Smith-Waterman algorithm, Appl. Math. 2:482-489; length = 56 or useful in searching sequence homology and Smith,T. F. and M. S. greater assembling DNA sequences. Waterman (1981) J.Mol. Biol. 147: 195-197; and Green, P., University of Washington,Seattle, WA. Consed A graphical tool for viewing and editing PhrapGordon, D. et al. (1998) assemblies. Genome Res. 8: 195-202. SPScan Aweight matrix analysis program that scans Nielson, H. et al. (1997)Score = 3.5 protein sequences for the presence of secretory ProteinEngineering 10: or greater signal peptides. 1-6; Claverie, J. M. and S.Audic (1997) CABIOS 12: 431-439. TMAP A program that uses weightmatrices to delineate Persson, B. and P. Argos transmembrane segments onprotein sequences and (1994) J. Mol. Biol. determine orientation. 237:182-192; Persson, B. and P. Argos (1996) Protein Sci. 5: 363-371.TMHMMER A program that uses a hidden Markov model (HMM) Sonnhammer, E.L. et al. to delineate transmembrane segments on protein (1998) Proc.Sixth sequences and determine orientation. Intl. Conf. On IntelligentSystems for Mol. Biol., Glasgow et al., eds., The Am. Assoc. forArtificial Intelligence (AAAI) Press, Menlo Park, CA, and MTT Press,Cambridge, MA, pp. 175-182. Motifs A program that searches amino acidsequences for Bairoch, A. et al. (1997) patterns that matched thosedefined in Prosite. Nucleic Acids Res. 25: 217-221; Wisconsin PackageProgram Manual, version 9, page M51-59, Genetics Computer Group,Madison, WI.

TABLE 8 SEQ EST All- All- Caucasian African Asian Hispanic ID EST CB1All- ele ele Amino Allele 1 Allele 1 Allele 1 Allele 1 NO: PID EST IDSNP ID SNP SNP ele 1 2 Acid frequency frequency frequency frequency 1537501111 3412087H1 SNP00120809 172 1481 T T G noncoding n/d n/a n/a n/a154 7501113 3412087H1 SNP00120809 172 1247 T T G noncoding n/d n/a n/an/a 155 7501118 3412087H1 SNP00120809 172 1199 T T G noncoding n/d n/an/a n/a 158 7510325 3412087H1 SNP00120809 172 2792 T T G noncoding n/dn/a n/a n/a 158 7510325 5439262H1 SNP00072289 114 2468 T T C noncodingn/a n/a n/a n/a 159 7510966 3412087H1 SNP00120809 172 1131 T T Gnoncoding n/d n/a n/a n/a

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-33, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 1-2, SEQ IDNO:4-13, SEQ ID NO: 15-19, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:28-29,and SEQ ID NO:31, c) a polypeptide comprising a naturally occurringamino acid sequence at least 93% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25, d)a polypeptide comprising a naturally occurring amino acid sequence atleast 95% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:22, and SEQ ID NO:27, e) apolypeptide comprising a naturally occurring amino acid sequence atleast 97% identical to the amino acid sequence of SEQ ID NO:30, f) apolypeptide comprising a naturally occurring amino acid sequence atleast 99% identical to the amino acid sequence of SEQ ID NO:33, g) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 1-33, and h)an immunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-33.
 2. An isolatedpolypeptide of claim 1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-33.
 3. An isolated polynucleotideencoding a polypeptide of claim
 1. 4. An isolated polynucleotideencoding a polypeptide of claim
 2. 5. An isolated polynucleotide ofclaim 4 comprising a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:34-66.
 6. A recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotide ofclaim
 3. 7. A cell transformed with a recombinant polynucleotide ofclaim
 6. 8. (CANCELLED)
 9. A method of producing a polypeptide of claim1, the method comprising: a) culturing a cell under conditions suitablefor expression of the polypeptide, wherein said cell is transformed witha recombinant polynucleotide, and said recombinant polynucleotidecomprises a promoter sequence operably linked to a polynucleotideencoding the polypeptide of claim 1, and b) recovering the polypeptideso expressed.
 10. A method of claim 9, wherein the polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:1-33.
 11. An isolated antibody which specifically binds to a polypeptideof claim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:34-66, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:34-56 and SEQ ID NO:58-66, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. (CANCELLED)
 14. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) hybridizingthe sample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 15. A methodof claim 14, wherein the probe comprises at least 60 contiguousnucleotides.
 16. A method of detecting a target polynucleotide in asample, said target polynucleotide having a sequence of a polynucleotideof claim 12, the method comprising: a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 1-33. 19.(CANCELLED)
 20. A method of screening a compound for effectiveness as anagonist of a polypeptide of claim 1, the method comprising: a) exposinga sample comprising a polypeptide of claim 1 to a compound, and b)detecting agonist activity in the sample. 21-22. (CANCELLED)
 23. Amethod of screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample. 24-25. (CANCELLED)
 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. (CANCELLED)
 28. A method of screening acompound for effectiveness in altering expression of a targetpolynucleotide, wherein said target polynucleotide comprises a sequenceof claim 5, the method comprising: a) exposing a sample comprising thetarget polynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method of assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.30.-112. (CANCELLED)